JP7582940B2 - Methods and devices for cell selection and stimulation - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62/753,911, entitled "METHODS FOR SELECTION AND STIMULATION OF CELLS AND KITS AND APPARATUS FOR SAME", filed October 31, 2018, U.S. Provisional Application No. 62/842,511, entitled "METHODS FOR SELECTION AND STIMULATION OF CELLS AND KITS AND APPARATUS FOR SAME", filed May 2, 2019, and U.S. Provisional Application No. 62/861,314, entitled "METHODS FOR SELECTION AND STIMULATION OF CELLS AND KITS AND APPARATUS FOR SAME", filed June 13, 2019, the contents of which are incorporated by reference in their entireties herein for all purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application is filed with an electronic Sequence Listing. The Sequence Listing is provided as a file named 7350423019240SeqList.txt, created on October 29, 2019, and is 94,112 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

Field The present disclosure provides a method for selecting and stimulating a plurality of cells in a cell sample using column chromatography, and collecting and/or eluting the cells without using additional steps or reagents to promote cell detachment from the column.In some aspects, the method provided herein reduces the time required to produce a population of selected and stimulated cells that are useful for genetic engineering and thus cell therapy compared to existing methods.The article of manufacture and device thereof are also provided.

Background A variety of cell therapy methods are available for treating diseases and conditions. Cell therapy methods include those using immune cells such as T cells (e.g., CD4+ and CD8+ T cells), which may be genetically engineered with recombinant receptors such as chimeric antigen receptors. Such methods for producing suitable cell populations for use in cell therapy, e.g., selected (enriched) and stimulated cell populations, often require separate selection and stimulation steps, which can lengthen the manufacturing process. Furthermore, selection techniques may involve contamination of selected cells with selection-related particles, e.g., selection agents such as Fab fragments, as well as competitive and/or free binding agents used to promote detachment of cells from the stationary phase, necessitating additional washing and/or medium changes to purify the output composition. Additional processing steps may result in cell stress, which in addition to taking a significant amount of time to complete, potentially impact downstream cell processing or even cell biology. Thus, there is a need for improved methods that minimize cell handling and processing times to produce cell populations suitable for use in, e.g., cell therapy. Methods, articles of manufacture and apparatus are provided that fulfill such needs.

Overview Provided is a method for on-column stimulation of T cells, comprising the steps of: adding an oligomeric stimulating reagent capable of delivering a stimulatory signal in a T cell to a stationary phase containing a plurality of immobilized T cells, thereby initiating incubation of the stimulating reagent with one or more T cells, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof, the oligomeric stimulating reagent comprising one or more stimulating agents comprising (i) a first stimulating agent that is an anti-CD3 antibody and (ii) a second stimulating agent that is an anti-CD28 antibody, and collecting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, thereby producing a composition containing stimulated T cells. In the provided methods, the selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof immobilizes the plurality of T cells onto the stationary phase.

A method for on-column stimulation of T cells is provided, comprising the steps of: incubating an oligomeric stimulating reagent capable of delivering a stimulatory signal in T cells with a plurality of immobilized T cells immobilized on a stationary phase, thereby initiating incubation of the stimulating reagent with one or more T cells, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof, the oligomeric stimulating reagent comprising one or more stimulating agents comprising (i) a first stimulating agent that is an anti-CD3 antibody and (ii) a second stimulating agent that is an anti-CD28 antibody, and collecting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, thereby producing a composition containing stimulated T cells. In the provided method, the selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof immobilizes the plurality of T cells on the stationary phase.

A method for on-column stimulation of T cells is provided, comprising the steps of: mixing an oligomeric stimulating reagent capable of delivering a stimulatory signal in T cells with a plurality of immobilized T cells immobilized on a stationary phase, thereby initiating incubation of the stimulating reagent with one or more T cells, the stationary phase comprising a selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof, the oligomeric stimulating reagent comprising one or more stimulating agents comprising (i) a first stimulating agent that is an anti-CD3 antibody and (ii) a second stimulating agent that is an anti-CD28 antibody; and collecting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, thereby producing a composition containing stimulated T cells. In the provided method, the selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof immobilizes the plurality of T cells on the stationary phase.

A method for on-column stimulation of T cells is provided, comprising the steps of: incubating a plurality of T cells immobilized on a stationary phase with one or more stimulatory agents to deliver a stimulatory signal in one or more T cells of the plurality of T cells, the stationary phase comprising a selection agent that specifically binds to a selection marker on the surface of the one or more T cells, wherein specific binding of the selection agent to the selection marker expressed by the one or more T cells results in immobilization of the one or more T cells to the stationary phase, and collecting the one or more T cells from the stationary phase by gravity flow within 24 hours from the start of incubation without adding a competitor or free binder to elute the plurality of T cells from the stationary phase, thereby producing a composition containing stimulated T cells. A method for on-column stimulation of T cells is provided, comprising: incubating a plurality of T cells immobilized on a stationary phase with one or more stimulatory substances to deliver a stimulatory signal in one or more T cells of the plurality of T cells, the stationary phase comprising a selection substance that specifically binds to a selection marker on the surface of the one or more T cells, the specific binding of the selection substance to the selection marker expressed by the one or more T cells immobilizing the one or more T cells on the stationary phase, and collecting the one or more T cells from the stationary phase by gravity flow within 24 hours from the start of incubation, thereby producing a composition containing stimulated T cells. In some embodiments, the stationary phase contains at least one of one or more stimulatory substances capable of delivering a stimulatory signal in the one or more T cells. In some embodiments, the at least one stimulatory substance is a first stimulatory substance, and the stationary phase further comprises one or more of a second stimulatory substance capable of enhancing, attenuating or modifying the stimulatory signal of the first stimulatory substance. In some embodiments, the stimulating agent is a first stimulating agent, and prior to the incubating step, a stimulating reagent containing one or more of the second stimulating agents capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating agent is added to the stationary phase. In some embodiments, the stimulating agent is a first stimulating agent, and prior to the incubating step, a stimulating reagent containing one or more of the second stimulating agents capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating agent is added to the stationary phase. In some embodiments, the one or more stimulating agents are a first stimulating agent and a second stimulating agent, and prior to the incubating step, a stimulating reagent containing a second stimulating agent capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating agent is added to the stationary phase. In some embodiments, prior to the incubating step, a stimulating reagent containing at least one of the one or more stimulating agents is added to the stationary phase.

In some embodiments, at least one of the stimulatory substances is a first stimulatory substance, and the one or more stimulatory substances further contain one or more of a second stimulatory substance capable of enhancing, attenuating or modifying the stimulatory signal of the first stimulatory substance. In some embodiments, at least one of the one or more stimulatory substances, optionally the first stimulatory substance, is capable of delivering a stimulatory signal, the stimulatory signal being a stimulatory signal via a TCR/CD3 complex in a T cell, a CD3-containing complex in a T cell, and/or an ITAM-containing molecule in a T cell. In some embodiments, at least one of the one or more first stimulatory substances is capable of delivering a stimulatory signal, the stimulatory signal being a stimulatory signal via a TCR/CD3 complex in a T cell, a CD3-containing complex in a T cell, and/or an ITAM-containing molecule in a T cell. In some embodiments, the second stimulatory substance is capable of specifically binding to a costimulatory molecule on the one or more T cells. In some embodiments, the first stimulatory agent delivers a stimulatory signal via the TCR/CD3 complex in the T cell, a CD3-containing complex in the T cell, and/or an ITAM-containing molecule in the T cell, and the second stimulatory agent binds to a costimulatory molecule on the T cell.

A method for on-column stimulation of T cells is provided, comprising the steps of: adding a sample containing a plurality of T cells to a stationary phase, the stationary phase containing a selection agent that binds to a selection marker on the surface of one or more of the plurality of T cells, thereby immobilizing the one or more of the plurality of T cells on the stationary phase; adding to the stationary phase a stimulating reagent containing one or more stimulating agents capable of delivering a stimulating signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and within 24 hours of initiating incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binding agent to elute the plurality of T cells from the stationary phase, thereby producing a composition containing stimulated T cells.

A method for on-column stimulation of T cells is provided, comprising the steps of: adding a sample containing a plurality of T cells to a stationary phase, the stationary phase containing a selection agent that binds to a selection marker on the surface of one or more of the plurality of T cells, thereby immobilizing the one or more of the plurality of T cells on the stationary phase; adding a stimulating reagent to the stationary phase, the stimulating reagent containing one or more stimulating agents capable of delivering a stimulatory signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and collecting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, thereby producing a composition containing stimulated T cells.

A method for on-column stimulation of T cells is provided, comprising the steps of: incubating a sample containing a plurality of T cells on a stationary phase, the stationary phase containing a selection agent that binds to a selection marker on the surface of one or more of the plurality of T cells, whereby the one or more of the plurality of T cells are immobilized on the stationary phase, with a stimulatory reagent containing one or more stimulatory agents capable of delivering a stimulatory signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulatory reagent with the one or more T cells; and within 24 hours of initiating incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binder to elute the plurality of T cells from the stationary phase, thereby producing a composition containing stimulated T cells.

A method for on-column stimulation of T cells is provided, comprising the steps of: (1) mixing (a) a sample containing a plurality of T cells with (b) a stationary phase containing a selection agent capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, whereby specific binding of the selection agent to the selection marker results in immobilization of the plurality of T cells to the stationary phase; (2) adding a stimulating reagent to the stationary phase containing one or more stimulating agents capable of delivering a stimulatory signal in the T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and (3) within 24 hours of initiating the incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binder to elute the plurality of T cells from the stationary phase, thereby producing a composition containing stimulated T cells.

A method for on-column stimulation of T cells is provided, the method comprising the steps of: (1) mixing (a) a sample containing a plurality of T cells with (b) a stationary phase containing a selection agent capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, whereby specific binding of the selection agent to the selection marker immobilizes the one or more of the plurality of T cells to the stationary phase; (2) adding a stimulating reagent to the stationary phase containing one or more stimulating agents capable of delivering a stimulatory signal in the T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and (3) collecting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating the incubation, thereby producing a composition containing stimulated T cells.

A method for on-column stimulation of T cells is provided, comprising the steps of: adding an oligomeric stimulating reagent to a stationary phase containing a plurality of immobilized T cells, thereby initiating incubation of the stimulating reagent with one or more T cells of the plurality of immobilized T cells, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells, and specific binding of the selection agent to the selection marker expressed by the one or more T cells results in immobilization of the one or more T cells to the stationary phase, wherein the oligomeric stimulating reagent contains (i) a plurality of streptavidin or streptavidin mutein molecules and (ii) one or more stimulating agents capable of delivering a stimulatory signal in one or more T cells, and the size of the oligomeric stimulating reagent comprises i) a radius of greater than 50 nm, ii) a molecular weight of at least 5×10 ⁶ g/mol, and/or (iii) at least 100 streptavidin or streptavidin mutein tetramers per oligomeric stimulating reagent. In some embodiments, the method further comprises harvesting one or more of said plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, thereby producing a composition containing stimulated T cells. In some embodiments, the method further comprises harvesting one or more of said plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, without adding a competitor or free binding agent to elute said plurality of T cells from the stationary phase, thereby producing a composition containing stimulated T cells.

In some embodiments, harvesting one or more of the plurality of T cells from the stationary phase occurs within about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours from the start of incubation. In some embodiments, harvesting one or more of the plurality of T cells from the stationary phase occurs within about 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, In some embodiments, the harvesting of one or more of the plurality of T cells from the stationary phase occurs within about 12, 10, 8, 6, 4, or 2 hours from the start of the incubation. In some embodiments, the harvesting of one or more of the plurality of T cells from the stationary phase occurs within 6 hours from the start of the incubation. In some embodiments, the harvesting of one or more of the plurality of T cells from the stationary phase occurs within 5 hours from the start of the incubation. In some embodiments, harvesting one or more of the plurality of T cells from the stationary phase occurs within or about 4.5 hours from the start of incubation. In some embodiments, harvesting one or more of the plurality of T cells from the stationary phase occurs within 4 hours from the start of incubation. In some embodiments, harvesting one or more of the plurality of T cells from the stationary phase occurs within 3 hours from the start of incubation.

In some embodiments, the initiation of incubation with the stimulating reagent is performed within or within 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within or within 20 minutes to 100 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within or within 30 minutes to 90 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 30 to 80 minutes or within about 30 to 80 minutes after adding or mixing the sample containing the plurality of T cells with the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 30 to 70 minutes or within about 30 to 70 minutes after adding or mixing the sample containing the plurality of T cells with the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 30 to 60 minutes or within about 30 to 60 minutes after adding or mixing the sample containing the plurality of T cells with the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 30 to 50 minutes or within about 30 to 50 minutes after adding or mixing the sample containing the plurality of T cells with the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 30 to 40 minutes or within about 30 to 40 minutes after adding or mixing the sample containing the plurality of T cells with the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 30 minutes or within about 30 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 40 minutes or within about 40 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 50 minutes or within about 50 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 60 minutes or within about 60 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within 70 minutes or within about 70 minutes after adding or mixing the sample containing the plurality of T cells to the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within or about 80 minutes after the sample containing the plurality of T cells is added to or mixed with the stationary phase. In some embodiments, the initiation of incubation with the stimulating reagent is performed within or about 90 minutes after the sample containing the plurality of T cells is added to or mixed with the stationary phase.

In some embodiments, at least one of the one or more stimulatory substances has the ability to deliver a stimulatory signal, the stimulatory signal being a stimulatory signal via the TCR/CD3 complex in the T cell, a CD3-containing complex in the T cell, and/or an ITAM-containing molecule in the T cell. In some embodiments, the at least one stimulatory substance is a first stimulatory substance, and the stimulatory reagent further contains one or more of a second stimulatory substance having the ability to enhance, attenuate, or modify the stimulatory signal of the first stimulatory substance. In some embodiments, the second stimulatory substance has the ability to specifically bind to a costimulatory molecule on the one or more T cells. In some embodiments, the costimulatory molecule is selected from among CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. In some embodiments, the second stimulatory substance has the ability to specifically bind to CD28. In some embodiments, the first stimulatory agent specifically binds to CD3 and the second stimulatory agent specifically binds to CD28.

In some embodiments, the stimuli are or contain a substance selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof; and/or the stimuli contain an antibody fragment; the stimuli are or contain a Fab fragment; the stimuli are selected from the group of bivalent antibody fragments consisting of (Fab)2' fragments and bivalent single chain Fv (scFv) fragments; the stimuli are monovalent antibody fragments selected from the group consisting of Fab fragments, Fv fragments and scFvs; and/or the stimuli are proteinaceous binding molecules with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystal scaffolds, adnectins and avimers.

In some embodiments, the first stimulatory substance and the second stimulatory substance are, or contain, independently, a substance selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and/or the first stimulatory substance and the second stimulatory substance are, or contain, an antibody fragment; the first stimulatory substance and the second stimulatory substance are, or contain, independently, a Fab fragment; the first stimulatory substance and the second stimulatory substance are, or contain, independently, a the first stimulatory agent and the second stimulatory agent are independently monovalent antibody fragments selected from the group consisting of Fab fragments, Fv fragments, and scFvs; and/or the first stimulatory agent and the second stimulatory agent are independently proteinaceous binding molecules with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, ankyrin scaffold-based proteins, crystal scaffold-based proteins, adnectins, and avimers.

In some embodiments, one or more stimulatory agents comprise a monovalent antibody fragment. In some embodiments, the first stimulatory agent and the second stimulatory agent independently comprise a monovalent antibody fragment. In some embodiments, the first stimulatory agent comprises a monovalent antibody fragment that binds CD3 and the second stimulatory agent comprises a monovalent antibody fragment that binds CD28. In some embodiments, the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv). In some embodiments, the first stimulatory agent is an anti-CD3 Fab and the second stimulatory agent is an anti-CD28 Fab.

A method for on-column stimulation of T cells is provided, comprising the steps of: adding an oligomeric stimulatory reagent capable of delivering a stimulatory signal in T cells to a stationary phase containing a plurality of immobilized T cells, thereby initiating incubation of the stimulatory reagent with one or more T cells, wherein the stationary phase contains a selection agent capable of specifically binding to a selection marker on the surface of one or more T cells or a subset thereof, wherein specific binding of the selection agent to the selection marker expressed by said one or more T cells or a subset thereof results in immobilization of said at least a plurality of T cells to the stationary phase, and wherein the selection agent and (iii) a second stimulatory agent, which is a Fab fragment capable of specifically binding to CD28 and has the ability to enhance, attenuate or modify the stimulatory signal, and the size of the oligomeric stimulatory agent is i) a radius of greater than 50 nm, ii) at least 5×10 and (iii) comprising at least ¹⁰⁰ streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulating reagent; and within 24 hours of initiating incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binding agent to elute the plurality of T cells from the stationary phase, thereby producing a composition containing stimulated T cells.

In some embodiments, the T cells are derived from a sample that is or includes a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product. In some embodiments, the sample is an apheresis product or a leukocyte apheresis product. In some embodiments, the apheresis product or leukocyte apheresis product has been previously cryofrozen.

In some embodiments, incubating with one or more stimuli releases one or more of the plurality of immobilized T cells from the stationary phase. In some embodiments, incubating with the first stimuli and the second stimuli releases one or more of the plurality of immobilized T cells from the stationary phase.

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグを、さらに含有する。いくつかの態様において、1つまたは複数の刺激物質は、

からなる群より選択されるストレプトアビジン結合ペプチドを、さらに含む。いくつかの態様において、1つまたは複数の刺激物質が、配列

を有するストレプトアビジン結合ペプチドを、さらに含む。 In some embodiments, the stimulator is biotin, a biotin analog that reversibly binds to streptavidin or avidin,

In some embodiments, the one or more stimuli further comprise a streptavidin-binding peptide selected from the group consisting of: a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.

In some embodiments, the one or more stimuli further comprise a streptavidin binding peptide selected from the group consisting of:

The present invention further includes a streptavidin binding peptide having the following structure:

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグを、さらに含有する。いくつかの態様において、第1刺激物質および第2刺激物質のそれぞれは、

からなる群より選択されるストレプトアビジン結合ペプチドを、さらに含む。いくつかの態様において、第1刺激物質および第2刺激物質のそれぞれは、配列

を有するストレプトアビジン結合ペプチドを、さらに含む。 In some embodiments, the first stimulatory agent and the second stimulatory agent are independently biotin, a biotin analog that reversibly binds to streptavidin or avidin,

In some embodiments, the first stimulatory agent and the second stimulatory agent further comprise a streptavidin-binding peptide selected from the group consisting of: a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.

In some embodiments, each of the first stimulatory agent and the second stimulatory agent further comprises a streptavidin-binding peptide selected from the group consisting of:

The present invention further includes a streptavidin binding peptide having the following structure:

In some embodiments, the selected agent is or contains an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like functions, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and/or the selected agent contains an antibody fragment; the selected agent is or contains a Fab fragment; the selected agent is selected from the group of bivalent antibody fragments consisting of (Fab)2' fragments and bivalent single chain Fv (scFv) fragments; the selected agent is a monovalent antibody fragment selected from the group consisting of Fab fragments, Fv fragments and scFvs; and/or the selected agent is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystal scaffolds, adnectins and avimers.

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグを、さらに含有する。いくつかの態様において、選択物質は、ビオチン、ストレプトアビジンまたはアビジンに可逆的に結合するビオチン類似体、

からなる群より選択されるストレプトアビジン結合ペプチドを、さらに含む。いくつかの態様において、選択物質は、配列

を有するストレプトアビジン結合ペプチドを、さらに含む。 In some embodiments, the selection agent is biotin, a biotin analogue that reversibly binds to streptavidin or avidin,

In some embodiments, the selection agent further comprises a streptavidin-binding peptide selected from the group consisting of: a calmodulin-binding peptide that reversibly binds to calmodulin; a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide; and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag. In some embodiments, the selection agent further comprises a biotin analog that reversibly binds to biotin, streptavidin, or avidin;

In some embodiments, the selection agent further comprises a streptavidin binding peptide selected from the group consisting of the sequence

The present invention further includes a streptavidin binding peptide having the following structure:

In some embodiments, the selection marker is a T cell coreceptor; the selection marker is or contains a member of the T cell antigen receptor complex; the selection marker is or contains a CD3 chain; the selection marker is or contains a CD3 zeta chain; the selection marker is or contains a CD8; the selection marker is or contains a CD4; the selection marker is or contains a CD45RA; the selection marker is or contains a CD27; the selection marker is or contains a CD28; and/or the selection marker is or contains a CCR7. In some embodiments, the selection marker is selected from the group consisting of CD3, CD4 and CD8. In some embodiments, the selection marker is CD3.

In some embodiments, the specific binding between the selection agent and the selection marker does not induce a signal to the T cell or does not induce a stimulatory, activating or proliferative signal to the T cell. In some embodiments, the selection agent comprises a monovalent antibody fragment that binds to CD3, CD8 or CD4. In some embodiments, the selection agent is an anti-CD3 Fab, an anti-CD8 Fab or an anti-CD4 Fab. In some embodiments, the selection agent is an anti-CD3 Fab. In some embodiments, the anti-CD3 Fab comprises an OKT3 antibody Fab fragment. In some embodiments, the anti-CD3 Fab comprises a variable heavy chain having the sequence shown in SEQ ID NO:31 and a variable light chain having the sequence shown in SEQ ID NO:32.

In some embodiments, the stimulating reagent is soluble. In some embodiments, the stimulating reagent is not, and is not bound to or associated with, a solid support, stationary phase, bead, microparticle, magnetic particle, and/or matrix; and/or the reagent is flexible, does not contain a metallic or magnetic core, is composed entirely or primarily of organic polymers, is not spherical, substantially spherical or uniform in shape, and/or is not rigid. In some embodiments, the stimulating reagent is or contains streptavidin, avidin, or a mutein of streptavidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof; avidin or a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide; a reagent containing at least two chelating groups K, the at least two chelating groups being capable of binding to a transition metal ion; a substance capable of binding to an oligohistidine affinity tag; a substance capable of binding to glutathione-S-transferase; calmodulin or an analog thereof; a substance capable of binding to calmodulin-binding peptide (CBP); a substance capable of binding to a FLAG peptide; a substance capable of binding to an HA tag; a substance capable of binding to maltose-binding protein (MBP); a substance capable of binding to an HSV epitope; a substance capable of binding to a myc epitope; or a substance capable of binding to a biotinylated carrier protein.

In some embodiments, the stimulating reagent is or contains a streptavidin or avidin mutein that reversibly binds to biotin or a biologically active fragment; the stimulating reagent is or contains a streptavidin or avidin mutein that reversibly binds to a biotin analog or a biologically active fragment; and/or the stimulating reagent is or contains a streptavidin or avidin mutein that reversibly binds to a streptavidin binding peptide.

In some embodiments, the stimulating reagent is an oligomeric stimulating reagent that contains multiple streptavidin molecules or streptavidin mutein molecules, and the size of the oligomeric stimulating particle reagent is i) a radius of more than 50 nm, ii) a molecular weight of at least 5×10 ⁶ g/mol, and/or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric particle reagent. In some embodiments, the oligomeric stimulating reagent is soluble. In some embodiments, the oligomeric stimulating reagent is not, and is not bound to or associated with, a solid support, a stationary phase, a bead, a microparticle, a magnetic particle, and/or a matrix; and/or the reagent is flexible, does not contain a metal core or a magnetic core, is composed entirely or mainly of organic polymers, and/or is not rigid.

からなる群より選択される。 In some embodiments, the streptavidin or streptavidin mutein molecule reversibly binds or has the ability to reversibly bind to biotin, a biotin analogue or a streptavidin binding peptide. In some embodiments, the streptavidin mutein contains the amino acid sequence Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 relative to the positions in streptavidin in the sequence of amino acids shown in SEQ ID NO:1, or the streptavidin mutein contains the amino acid sequence Val44-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 relative to the positions in streptavidin in the sequence of amino acids shown in SEQ ID NO:1. In some embodiments, the streptavidin binding peptide is

is selected from the group consisting of:

In some embodiments, the oligomeric particle reagent comprises a radius of greater than 60 nm, greater than 70 nm, greater than 80 nm, or greater than 90 nm. In some embodiments, the oligomeric particle reagent comprises a radius of 50 nm to 150 nm, 75 nm to 125 nm, 80 nm to 115 nm, or 90 nm to 110 nm, inclusive, or a radius of 90 nm ± 15 nm or 95 nm ± 20 to 25 nm. In some embodiments, the radius is the hydrodynamic radius.

In some embodiments, the oligomeric particle reagent comprises the following molecular weights: at least 5×10 ⁷ g/mol or at least 1×10 ⁸ g/mol; and/or 5×10 ⁷ g/mol to 5×10 ⁸ g/mol, 1×10 ⁸ g/mol to 5×10 ⁸ g/mol, or 1×10 ⁸ g/mol to 2×10 ⁸ g/mol. In some embodiments, the oligomeric particle reagent contains at least 500 streptavidin tetramers or streptavidin mutein tetramers, at least 1,000 streptavidin tetramers or streptavidin mutein tetramers, at least 1,500 streptavidin tetramers or streptavidin mutein tetramers, or at least 2,000 streptavidin tetramers or streptavidin mutein tetramers; and/or 1,000-20,000 streptavidin tetramers or streptavidin mutein tetramers, 1,000-10,000 streptavidin tetramers or streptavidin mutein tetramers, or 2,000-5,000 streptavidin tetramers or streptavidin mutein tetramers. In some embodiments, the oligomeric stimulation reagent is added to the stationary phase at a concentration of about 1 to about 2 μg/million cells.

In some embodiments, the selection agent is directly or indirectly bound to the stationary phase. In some embodiments, the selection agent is indirectly bound to the stationary phase via a selection reagent to which the selection agent reversibly binds. In some embodiments, the selection reagent is or contains streptavidin, avidin, or a mutein of streptavidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof; an avidin or mutein of streptavidin that reversibly binds to a streptavidin-binding peptide; a reagent containing at least two chelating groups K, said at least two chelating groups being capable of binding to a transition metal ion; a substance capable of binding to an oligohistidine affinity tag; a substance capable of binding to glutathione-S-transferase; calmodulin or an analog thereof; a substance capable of binding to calmodulin-binding peptide (CBP); a substance capable of binding to a FLAG peptide; a substance capable of binding to an HA tag; a substance capable of binding to maltose binding protein (MBP); a substance capable of binding to an HSV epitope; a substance capable of binding to a myc epitope; or a substance capable of binding to a biotinylated carrier protein. In some embodiments, the selection reagent is or contains a streptavidin or avidin mutein that reversibly binds to biotin or a biologically active fragment; the stimulating reagent is or contains a streptavidin or avidin mutein that reversibly binds to a biotin analog or a biologically active fragment; and/or the stimulating reagent is or contains a streptavidin or avidin mutein that reversibly binds to a streptavidin binding peptide. In some embodiments, the streptavidin or streptavidin mutein molecule reversibly binds or has the ability to reversibly bind to biotin, a biotin analog, or a streptavidin binding peptide.

からなる群より選択される。いくつかの態様において、ストレプトアビジン結合ペプチドは、配列

を有する。 In some embodiments, the streptavidin mutein contains the amino acid sequence Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 in streptavidin relative to the positions in streptavidin in the sequence of amino acids shown in SEQ ID NO:1, or the streptavidin mutein contains the amino acid sequence Val44-Thr45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 in streptavidin relative to the positions in streptavidin in the sequence of amino acids shown in SEQ ID NO:1.

In some embodiments, the streptavidin binding peptide is selected from the group consisting of the sequence

has.

In some embodiments, the method provided herein is carried out at or about 37°C. In some embodiments, the harvesting includes washing the stationary phase with a medium that does not contain a competitor or free binder for eluting the T cells from the stationary phase. In some embodiments, the harvesting by gravity flow includes adding to the stationary phase a medium that does not contain a competitor or free binder for eluting the T cells from the stationary phase. In some embodiments, the composition containing the stimulated T cells does not contain a competitor or free binder. In some embodiments, the competitor or free binder is or contains biotin or a biotin analog, and optionally, the biotin analog is D-biotin. In some embodiments, the competitor or free binder is D-biotin. In some embodiments, the method includes further incubating the composition containing the stimulated T cells after the harvesting. In some embodiments, the further incubation is carried out at or about 37°C±2°C; and/or the further incubation is carried out in the presence of an additional agent capable of delivering a signal in the T cells. In some embodiments, the additional agent is contained in the medium used to wash the stationary phase. In some embodiments, the additional agent has the ability to enhance or induce proliferation of T cells, CD4+ T cells and/or CD8+ T cells. In some embodiments, the additional agent is a cytokine selected from among IL-2, IL-15 and IL-7. In some embodiments, the additional incubation is performed for a period of 72 hours, 48 hours or less, 24 hours or less, or 12 hours or less.

In some embodiments, the method further comprises introducing a recombinant nucleic acid molecule encoding a recombinant protein into the stimulated T cells of the composition, thereby producing a composition comprising the transduced T cells. In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a chimeric antigen receptor.

In some embodiments, the chimeric antigen receptor (CAR) contains an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain that includes an ITAM. In some embodiments, the intracellular signaling domain includes the intracellular domain of the CD3-zeta (CD3ζ) chain. In some embodiments, a transmembrane domain is further included that links the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain includes the transmembrane portion of CD28. In some embodiments, the intracellular signaling domain further includes an intracellular signaling domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB. In some embodiments, the nucleic acid further includes a promoter operably linked to the nucleic acid encoding the recombinant antigen receptor.

In some embodiments, the introduction of the recombinant nucleic acid is accomplished by transduction with a viral particle. In some embodiments, the viral particle is a retroviral vector particle. In some embodiments, the viral particle is a lentiviral vector particle.

In some embodiments, the method further comprises incubating the composition comprising the transduced cells under conditions for viral integration, optionally at or about 37°±2° C. In some embodiments, the step of incubating the composition comprising the transduced cells is performed for up to 96 hours after introduction. In some embodiments, the step of incubating the composition comprising the transduced cells is performed for up to 72 hours after introduction. In some embodiments, the step of incubating the composition comprising the transduced cells is performed for up to 48 hours after introduction. In some embodiments, the step of incubating the composition comprising the transduced cells is performed for up to 24 hours after introduction. In some embodiments, the step of incubating the composition comprising the transduced cells is performed for at least 18 hours after introduction.

In some embodiments, the method further comprises culturing the composition containing the transduced cells under conditions for viral integration, thereby producing a composition containing cultured T cells. In some embodiments, the method further comprises culturing the composition containing the transduced cells under conditions for expanding the T cells. In some embodiments, the culturing is carried out for a period of 14 days or less, 12 days or less, 10 days or less, 8 days or less, or 6 days or less. In some embodiments, for 5 days or less.

In some embodiments, the method further comprises harvesting the engineered T cells, thereby generating an output population of engineered T cells. In some embodiments, the method further comprises harvesting the engineered T cells at 48 to 120 hours, inclusive, after initiating exposure to the stimulatory agent. In some embodiments, the harvesting is performed within 120 hours after initiating exposure to the stimulatory agent. In some embodiments, the harvesting is performed within 96 hours after initiating exposure to the stimulatory agent. In some embodiments, the harvesting is performed within 72 hours after initiating exposure to the stimulatory agent. In some embodiments, the harvesting is performed within 48 hours after initiating exposure to the stimulatory agent.

In some embodiments, the percentage of naive-like cells is greater than or greater than about 60% of the total T cells in the population, the total CD4+ T cells in the population, or the CD8+ T cells or recombinant protein-expressing cells thereof in the population at the time of harvest. In some embodiments, the naive-like T cells comprise CD27+CCR7+ cells.

In some embodiments, the introducing is performed in a serum-free medium. In some embodiments, the incubating step is performed in a serum-free medium. In some embodiments, the culturing step is performed in a serum-free medium. In some embodiments, the serum-free medium contains 0.5 mM to 5 mM dipeptide form of L-glutamine, 0.5 mM to 5 mM L-glutamine, and optionally at least one protein in a basal medium, the medium being serum-free. In some embodiments, the serum-free medium contains a recombinant cytokine selected from among IL-2, IL-15, and IL-7, optionally recombinant human IL-2, recombinant human IL-15, and/or recombinant human IL-7. In some embodiments, the serum-free medium does not contain a recombinant cytokine selected from among IL-2, IL-15, and IL-7, optionally recombinant human IL-2, recombinant human IL-15, and/or recombinant human IL-7.

In some embodiments, the method further comprises incubating the composition containing the transduced cells. In some embodiments, the incubation is carried out for at or about 24 hours ± 6 hours, 48 hours ± 6 hours, or 72 hours ± 6 hours.

In some embodiments, the method further comprises adding a competitor or free binder to a composition containing stimulated T cells, thereby disrupting said reversible binding. In some embodiments, the method further comprises adding a competitor or free binder to a composition containing transduced T cells, thereby disrupting said reversible binding. In some embodiments, the method further comprises adding a competitor or free binder to a composition containing cultured T cells, thereby disrupting said reversible binding. In some embodiments, the method further comprises adding a competitor or free binder to a composition containing engineered cells, optionally transduced T cells, optionally under conditions to dissociate one or more stimulatory agents from the oligomeric stimulating reagent in the composition. In some embodiments, the method further comprises adding a competitor or free binder to a composition containing incubated T cells, optionally under conditions to dissociate one or more stimulatory agents from the oligomeric stimulating reagent in the composition. In some embodiments, the method further comprises adding a competitor or free binder to the composition containing the cultured T cells, optionally under conditions to dissociate one or more stimulatory agents from the oligomeric stimulating reagent in the composition. In some embodiments, the step of adding the competitor or free binder is performed prior to harvesting.

In some embodiments, the competitor or free binder is not detrimental to the T cells and/or addition of the substance does not reduce the percentage of surviving T cells to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% compared to incubation of T cells under equivalent or identical conditions without the competitor or free binder. In some embodiments, the disruption terminates or reduces the stimulatory signal in the T cells. In some embodiments, the competing reagent and the free binding agent independently comprise a molecule from the group consisting of a streptavidin binding molecule; biotin; D-biotin; a biotin analog; a biotin analog that specifically binds to streptavidin or a streptavidin analog having the amino acid sequence Val44-Thr45-Ala46-Arg47 or Ile44-Gly45-Ala46-Arg47 at sequence positions corresponding to positions 44-47 of wild-type streptavidin; or the competing reagent and the free binding agent independently comprise a metal chelator, optionally EDTA or EGTA. In some embodiments, the competing agent or the free binding agent is D-biotin, optionally 1 mM D-biotin. In some embodiments, the method further comprises washing the cells, optionally, the washing step reduces or removes the stimulating reagent and/or the one or more stimuli in the composition. In some embodiments, the washing step is performed prior to harvesting.

In some embodiments, the T cells comprise antigen-specific T cells or populations thereof, T helper cells or populations thereof, cytotoxic T cells or populations thereof, memory T cells or populations thereof, or regulatory T cells or populations thereof. In some embodiments, the T cells comprise CD3+ T cells, or comprise CD4+ and/or CD8+ T cells.

In some embodiments, the method includes selecting a T cell subset from the stimulated T cells of the composition, followed by an introducing step, where the recombinant nucleic acid molecule is introduced into the selected T cell subset. In some embodiments, the method includes selecting a T cell subset from a composition containing transduced cells, followed by an incubation step, where the selected T cell subset is incubated under conditions for viral integration. In some embodiments, the method includes selecting a T cell subset from a composition containing engineered cells, followed by a culturing step, where the selected T cell subset is cultured under conditions for expanding the T cells. In some embodiments, the method includes selecting a T cell subset from a composition containing engineered cells, followed by a harvesting step, where the selected T cell subset is harvested to create an output population of engineered T cells. In some embodiments, the T cell subset is a naive-like T cell, or the T cells that are surface positive for a marker expressed on a naive-like T cell are CCR7+CD45RA+, CD27+CCR7+, or CD62L-CCR7+. In some embodiments, the naive-like T cells comprise CD27+CCR7+ T cells. In some embodiments, the naive-like T cells comprise CCR7+CD45RA+ T cells. In some embodiments, the T cell subset expresses a recombinant protein, optionally a chimeric antigen receptor. In some embodiments, the step of selecting the T cell subset is performed by affinity column chromatography.

In some embodiments, the method further comprises formulating the harvested cells, optionally in the presence of a pharma- ceutically acceptable excipient, for cryopreservation and/or administration to a subject. In some embodiments, the harvested cells are formulated in the presence of a cryoprotectant. In some embodiments.

In some embodiments, the stationary phase is or comprises a chromatographic matrix. In some embodiments, the stationary phase has a binding capacity, optionally a static or dynamic binding capacity, of about 75 million to about 125 million T cells per mL of stationary phase. In some embodiments, (a) the stationary phase is about 20 mL; and/or (b) the stationary phase has a binding capacity of 2 billion ± 500 million cells. In some embodiments, the method comprises two stationary phases. In some embodiments, the two stationary phases are arranged in parallel. In some embodiments, the two stationary phases are arranged sequentially.

An article of manufacture for on-column stimulation of T cells is provided, comprising a first stimulatory substance and a second stimulatory substance capable of stimulating a T cell by specifically binding to a first molecule and a second molecule, respectively, on the surface of the T cell, and a stationary phase comprising a selection substance capable of immobilizing a T cell on the stationary phase by specifically binding to a selection marker on the T cell. In some embodiments, the stationary phase further comprises the first stimulatory substance and the second stimulatory substance. In some embodiments, the first stimulatory substance, the second stimulatory substance and the selection substance are indirectly bound to the stationary phase via a selection reagent. In some embodiments, the article further comprises a stimulatory reagent to which the first stimulatory substance and the second stimulatory substance are reversibly bound or can be reversibly bound. In some embodiments, the stimulatory reagent is an oligomeric stimulatory reagent. In some embodiments, the selection substance is indirectly bound to the stationary phase via a selection reagent.

In some embodiments, the stationary phase is or includes a chromatography matrix, and the article of manufacture further includes a container that contains all or a portion of the chromatography matrix. In some embodiments, the article of manufacture includes two stationary phases. In some embodiments, the two stationary phases are arranged in parallel. In some embodiments, the two stationary phases are arranged sequentially.

Apparatuses including the articles of manufacture and embodiments thereof are provided. In some embodiments, the apparatus further includes a fluid inlet fluidly connected to one or more components of the apparatus and/or a fluid outlet fluidly connected to one or more components of the apparatus. In some embodiments, the apparatus is in a closed system or a sterile system. In some embodiments, the system is both a closed and a sterile system.

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグをさらに含む、本発明1001～1027、1029および1035～1028のいずれかの方法。
[本発明1041]
1つまたは複数の刺激物質が、

からなる群より選択されるストレプトアビジン結合ペプチドをさらに含む、本発明1001～1027、1029および1035～1040のいずれかの方法。
[本発明1042]
1つまたは複数の刺激物質が、配列

を有するストレプトアビジン結合ペプチドをさらに含む、本発明1001～1027、1029および1035～1041のいずれかの方法。
[本発明1043]
第1刺激物質および第2刺激物質が、独立して、ビオチン、ストレプトアビジンまたはアビジンに可逆的に結合するビオチン類似体、

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグをさらに含む、本発明1001、1004、1006、1007、1008、1013～1020、1022～1026、1028、1030～1039のいずれかの方法。
[本発明1044]
第1刺激物質および第2刺激物質のそれぞれが、

からなる群より選択されるストレプトアビジン結合ペプチドをさらに含む、本発明1001、1004、1006、1007、1008、1013～1020、1022～1026、1028、1030～1039および1043のいずれかの方法。
[本発明1045]
第1刺激物質および第2刺激物質のそれぞれが、配列

を有するストレプトアビジン結合ペプチドをさらに含む、本発明1001、1004、1006、1007、1008、1013～1020、1022～1026、1028、1030～1039、1043および1044のいずれかの方法。
[本発明1046]
選択物質が、抗体フラグメント、一価抗体フラグメント、免疫グロブリン様機能を持つタンパク質性結合分子、Igドメインを含有する分子、サイトカイン、ケモカイン、アプタマー、MHC分子、MHC-ペプチド複合体;受容体リガンド;およびその結合性フラグメントからなる群より選択される物質であるか、またはそれを含む、本発明1001～1045のいずれかの方法。
[本発明1047]
選択マーカーがT細胞補助受容体であり;
選択マーカーがT細胞抗原受容体複合体のメンバーであるか、もしくはそれを含み、
選択マーカーがCD3鎖であるか、もしくはそれを含み、
選択マーカーがCD3ゼータ鎖であるか、もしくはそれを含み、
選択マーカーがCD8であるか、もしくはそれを含み;
選択マーカーがCD4であるか、もしくはそれを含み、
選択マーカーがCD45RAであるか、もしくはそれを含み;
選択マーカーがCD27であるか、もしくはそれを含み、
選択マーカーがCD28であるか、もしくはそれを含み、および/または
選択マーカーがCCR7であるか、もしくはそれを含む、
本発明1001～1046のいずれかの方法。
[本発明1048]
選択マーカーが、CD3、CD4およびCD8からなる群より選択される、本発明1001～1047のいずれかの方法。
[本発明1049]
選択マーカーがCD3である、本発明1001～1048のいずれかの方法。
[本発明1050]
選択物質が、ビオチン、ストレプトアビジンまたはアビジンに可逆的に結合するビオチン類似体、

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグをさらに含む、本発明1001～1049のいずれかの方法。
[本発明1051]
選択物質が、ビオチン、ストレプトアビジンまたはアビジンに可逆的に結合するビオチン類似体、

からなる群より選択されるストレプトアビジン結合ペプチドをさらに含む、本発明1001～1050のいずれかの方法。
[本発明1052]
選択物質が、配列

を有するストレプトアビジン結合ペプチドをさらに含む、本発明1001～1051のいずれかの方法。
[本発明1053]
選択物質と選択マーカーの間の特異的結合が、T細胞へのシグナルを誘導しないか、または、T細胞への刺激シグナル、活性化シグナルおよび増殖シグナルをいずれも誘導しない、本発明1001～1052のいずれかの方法。
[本発明1054]
選択物質が、CD3、CD8またはCD4に結合する一価抗体フラグメントを含む、本発明1001～1053のいずれかの方法。
[本発明1055]
選択物質が抗CD3 Fab、抗CD8 Fabまたは抗CD4 Fabである、本発明1001～1054のいずれかの方法。
[本発明1056]
選択物質が抗CD3 Fabである、本発明1001～1055のいずれかの方法。
[本発明1057]
刺激試薬が、複数のストレプトアビジン分子またはストレプトアビジンムテイン分子を含むオリゴマー刺激試薬であり、該オリゴマー粒子試薬のサイズが、（i）50nm超の半径、（ii）少なくとも5×10 ⁶ g/molの分子量および/または（iii）オリゴマー刺激試薬あたり少なくとも100個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体を含む、本発明1004～1010、1013～1033および1035～1056のいずれかの方法。
[本発明1058]
オリゴマー刺激試薬が、複数のストレプトアビジン分子またはストレプトアビジンムテイン分子を含み、該オリゴマー粒子試薬のサイズが、（i）50nm超の半径、（ii）少なくとも5×10 ⁶ g/molの分子量および/または（iii）オリゴマー刺激試薬あたり少なくとも100個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体を含む、本発明1001の方法。
[本発明1059]
オリゴマー刺激試薬が可溶性である、本発明1001、1011～1058のいずれかの方法。
[本発明1060]
オリゴマー刺激試薬が、固形支持体、固定相、ビーズ、微粒子、磁気粒子および/もしくはマトリックスではなく、かつそれらに結合も会合もしておらず、ならびに/または
該試薬が、フレキシブルであり、金属コアも磁気コアも含有せず、完全にもしくは主として有機多量体で構成され、かつ/もしくは剛直ではない、
本発明1001、1011～1059のいずれかの方法。
[本発明1061]
ストレプトアビジン分子またはストレプトアビジンムテイン分子が、ビオチン、ビオチン類似体もしくはストレプトアビジン結合ペプチドに可逆的に結合するか、または可逆的に結合する能力を有する、本発明1011～1060のいずれかの方法。
[本発明1062]
ストレプトアビジンムテインが、SEQ ID NO:1に示すアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ を含むか、または
ストレプトアビジンムテインが、SEQ ID NO:1に示すアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ を含む、
本発明1011～1061のいずれかの方法。
[本発明1063]
ストレプトアビジン結合ペプチドが、

からなる群より選択される、本発明1061または1062のいずれかの方法。
[本発明1064]
オリゴマー刺激試薬が、
60nm超、70nm超、80nm超または90nm超の半径
を含む、本発明1001および1011～1063のいずれかの方法。
[本発明1065]
オリゴマー刺激試薬が、
両端の値を含む50nm～150nm、75nm～125nm、80nm～115nmもしくは90nm～110nmの半径、または
90nm±15nmもしくは95nm±20～25nmの半径
を含む、本発明1001および1011～1064のいずれかの方法。
[本発明1066]
前記半径が流体力学的半径である、本発明1164のいずれかの方法。
[本発明1067]
オリゴマー刺激試薬が、
少なくとも5×10 ⁷ g/molもしくは少なくとも1×10 ⁸ g/mol、および/または
5×10 ⁷ g/mol～5×10 ⁸ g/mol、1×10 ⁸ g/mol～5×10 ⁸ g/mol、もしくは1×10 ⁸ g/mol～2×10 ⁸ g/mol
の分子量を含む、本発明1001および1011～1066のいずれかの方法。
[本発明1068]
オリゴマー刺激試薬が、
少なくとも500個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、少なくとも1,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、少なくとも1,500個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、または少なくとも2,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、および/または
1,000～20,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、1,000～10,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、または2,000～5,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体
を含む、本発明1001および1011～1067のいずれかの方法。
[本発明1069]
オリゴマー刺激試薬が、約1～約2μg/細胞100万個の濃度で固定相に加えられる、本発明1001および1011～1068のいずれかの方法。
[本発明1070]
選択物質が固定相に直接的または間接的に結合される、本発明1001～1069のいずれかの方法。
[本発明1071]
選択物質が、該選択物質が可逆的に結合する選択試薬を介して間接的に固定相に結合される、本発明1001～1070のいずれかの方法。
[本発明1072]
選択試薬が、ストレプトアビジン;アビジン;ビオチン、ビオチン類似体もしくは生物学的に活性なそのフラグメントに可逆的に結合するストレプトアビジンのムテイン;ストレプトアビジン結合ペプチドに可逆的に結合する、アビジンもしくはストレプトアビジンのムテイン;少なくとも2つのキレート基Kを含む試薬であって、該少なくとも2つのキレート基が、遷移金属イオンに結合する能力を有する、試薬;オリゴヒスチジンアフィニティータグに結合する能力を有する物質;グルタチオン-S-トランスフェラーゼに結合する能力を有する物質;カルモジュリンまたはその類似体;カルモジュリン結合ペプチド（CBP）に結合する能力を有する物質;FLAGペプチドに結合する能力を有する物質;HAタグに結合する能力を有する物質;マルトース結合タンパク質（MBP）に結合する能力を有する物質;HSVエピトープに結合する能力を有する物質;mycエピトープに結合する能力を有する物質;またはビオチン化担体タンパク質に結合する能力を有する物質であるか、またはそれを含む、本発明1070または本発明1071の方法。
[本発明1073]
選択試薬が、ビオチンもしくは生物学的に活性なフラグメントに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含み、
刺激試薬が、ビオチン類似体もしくは生物学的に活性なフラグメントに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含み、および/あるいは
刺激試薬が、ストレプトアビジン結合ペプチドに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含む、
本発明1070～1072のいずれかの方法。
[本発明1074]
ストレプトアビジン分子またはストレプトアビジンムテイン分子が、ビオチン、ビオチン類似体もしくはストレプトアビジン結合ペプチドに可逆的に結合するか、または可逆的に結合する能力を有する、本発明1072または本発明1073の方法。
[本発明1075]
ストレプトアビジンムテインが、SEQ ID NO:1に示すアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ を含むか、または
ストレプトアビジンムテインが、SEQ ID NO:1に示すアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ を含む、
本発明1072～1074のいずれかの方法。
[本発明1076]
ストレプトアビジン結合ペプチドが、

からなる群より選択される、本発明1072～1075のいずれかの方法。
[本発明1077]
ストレプトアビジン結合ペプチドが、配列

を有する、本発明1072～1076のいずれかの方法。
[本発明1078]
重力流によって収集することが、固定相からT細胞を溶出させるための競合剤も遊離結合剤（free binding agent）も含まない培地を固定相に加えることを含む、本発明1001～1010または1012～1077のいずれかの方法。
[本発明1079]
刺激されたT細胞を含む組成物が競合剤も遊離結合剤も含まない、本発明1001～1010および1012～1078のいずれかの方法。
[本発明1080]
競合剤または遊離結合剤が、ビオチンもしくはビオチン類似体であるか、またはそれを含み、任意で、該ビオチン類似体が、D-ビオチンである、本発明1078または1079の方法。
[本発明1081]
前記組成物の刺激されたT細胞に、組換えタンパク質をコードする組換え核酸分子を導入し、それによって、操作されたT細胞を含む組成物、任意で形質導入T細胞を含む組成物を作製する工程をさらに含む、本発明1001～1078のいずれかの方法。
[本発明1082]
組換えタンパク質が抗原受容体である、本発明1081の方法。
[本発明1083]
組換えタンパク質がキメラ抗原受容体である、本発明1081または本発明1082の方法。
[本発明1084]
キメラ抗原受容体（CAR）が、標的抗原に特異的に結合する細胞外抗原認識ドメインと、ITAMを含む細胞内シグナル伝達ドメインとを含む、本発明1083の方法。
[本発明1085]
細胞内シグナル伝達ドメインが、CD3ゼータ（CD3ζ）鎖の細胞内ドメインを含む、本発明1084の方法。
[本発明1086]
細胞外ドメインと細胞内シグナル伝達ドメインとを連結する膜貫通ドメインをさらに含む、本発明1084または本発明1085の方法。
[本発明1087]
膜貫通ドメインがCD28の膜貫通部分を含む、本発明1086の方法。
[本発明1088]
細胞内シグナル伝達ドメインがT細胞共刺激分子の細胞内シグナル伝達ドメインをさらに含む、本発明1084～1087のいずれかの方法。
[本発明1089]
T細胞共刺激分子が、CD28および41BBからなる群より選択される、本発明1088の方法。
[本発明1090]
前記核酸が、組換え抗原受容体をコードする核酸に機能的に連結されたプロモータをさらに含む、本発明1081～1089のいずれかの方法。
[本発明1091]
組換え核酸の導入が、ウイルス粒子を使った形質導入によって達成される、本発明1081～1090のいずれかの方法。
[本発明1092]
ウイルス粒子がレトロウイルスベクター粒子である、本発明1091の方法。
[本発明1093]
ウイルス粒子がレンチウイルスベクター粒子である、本発明1091または本発明1092の方法。
[本発明1094]
形質導入細胞を含む組成物を、ウイルス組込みのための条件下、任意で37°±2℃または約37°±2℃の温度で、インキュベートする工程をさらに含む、本発明1081～1093のいずれかの方法。
[本発明1095]
形質導入細胞を含む組成物をインキュベートする工程が、導入後、最大96時間にわたって実行される、本発明1094の方法。
[本発明1096]
形質導入細胞を含む組成物をインキュベートする工程が、導入後、最大72時間にわたって実行される、本発明1094の方法。
[本発明1097]
形質導入細胞を含む組成物をインキュベートする工程が、導入後、最大48時間にわたって実行される、本発明1094の方法。
[本発明1098]
形質導入細胞を含む組成物をインキュベートする工程が、導入後、最大24時間にわたって実行される、本発明1094の方法。
[本発明1099]
形質導入細胞を含む組成物をインキュベートする工程が、導入後、少なくとも18時間にわたって実行される、本発明1094～1098のいずれかの方法。
[本発明1100]
操作された細胞を含む組成物、任意で形質導入細胞を含む組成物を、T細胞を拡大培養するための条件下で培養する工程をさらに含む、本発明1081～1099のいずれかの方法。
[本発明1101]
培養する工程が14日以下、12日以下、10日以下、8日以下、6日以下または5日以下の時間にわたって実行される、本発明1100の方法。
[本発明1102]
操作されたT細胞を採取し、それによって、操作されたT細胞のアウトプット集団を作製する工程をさらに含む、本発明1081～1101のいずれかの方法。
[本発明1103]
刺激試薬への曝露を開始した後、両端の値を含む48～120時間の時点で、操作されたT細胞を採取する工程をさらに含む、本発明1081～1099のいずれかの方法。
[本発明1104]
刺激物質への曝露を開始した後、120時間以内に採取が実行される、本発明1081～1099および1103のいずれかの方法。
[本発明1105]
刺激物質への曝露を開始した後、96時間以内に採取が実行される、本発明1081～1099および1103のいずれかの方法。
[本発明1106]
刺激物質への曝露を開始した後、72時間以内に採取が実行される、本発明1081～1099および1105のいずれかの方法。
[本発明1107]
刺激物質への曝露を開始した後、48時間以内に採取が実行される、本発明1081～1099および1106のいずれかの方法。
[本発明1108]
ナイーブ様細胞のパーセンテージが、採取の時点で、集団中の全T細胞、集団中の全CD4＋T細胞、または集団中の全CD8＋T細胞もしくはその組換えタンパク質発現細胞のうちの60％超または約60％超である、本発明1102～1107のいずれかの方法。
[本発明1109]
ナイーブ様T細胞がCD27＋CCR7＋細胞を含む、本発明1108の方法。
[本発明1110]
導入が無血清培地中で実行される、本発明1081～1093のいずれかの方法。
[本発明1111]
インキュベートする工程が無血清培地中で実行される、本発明1094～1099のいずれかの方法。
[本発明1112]
培養する工程が無血清培地中で実行される、本発明1100または本発明1101の方法。
[本発明1113]
無血清培地が、
基本培地中、0.5mM～5mMのジペプチド型L-グルタミン、
0.5mM～5mM L-グルタミン、および
任意で少なくとも1つのタンパク質
を含み、該培地が血清を含まない、本発明1110～1112のいずれかの方法。
[本発明1114]
無血清培地が、IL-2、IL-15およびIL-7の中から選択される組換えサイトカイン、任意で、組換えヒトIL-2、組換えヒトIL-15および/または組換えヒトIL-7を含む、本発明1110～1113のいずれかの方法。
[本発明1115]
無血清培地が、IL-2、IL-15およびIL-7の中から選択される組換えサイトカイン、任意で、組換えヒトIL-2、組換えヒトIL-15および/または組換えヒトIL-7を含まない、本発明1110～1114のいずれかの方法。
[本発明1116]
操作された細胞を含む組成物、任意で形質導入T細胞を含む組成物に競合剤または遊離結合剤を加える工程をさらに含み、任意で、該剤が、1つまたは複数の刺激物質を該組成物中のオリゴマー刺激試薬から解離させるための条件下で加えられる、本発明1081～1093のいずれかの方法。
[本発明1117]
インキュベートされたT細胞を含む組成物に、任意で、1つまたは複数の刺激物質を該組成物中のオリゴマー刺激試薬から解離させるための条件下で、競合剤または遊離結合剤を加える工程をさらに含む、本発明1094～1099のいずれかの方法。
[本発明1118]
培養されたT細胞を含む組成物に、任意で、1つまたは複数の刺激物質を該組成物中のオリゴマー刺激試薬から解離させるための条件下で、競合剤または遊離結合剤を加える工程をさらに含む、本発明1100～1101のいずれかの方法。
[本発明1119]
競合剤または遊離結合剤を添加する工程が、採取に先立って実行される、本発明1116～1118のいずれかの方法。
[本発明1120]
競合剤もしくは遊離結合剤が、T細胞にとって有害ではなく、および/または、該物質の添加が、該競合剤も該遊離結合剤も使用しない同等もしくは同じ条件下でのT細胞のインキュベーションとの比較で、生残するT細胞のパーセンテージを90％未満、80％未満、70％未満、60％未満もしくは50％未満に低減させることはない、本発明1116～1119のいずれかの方法。
[本発明1121]
前記解離が、T細胞における刺激シグナルを終結または軽減させる、本発明1116～1120のいずれかの方法。
[本発明1122]
競合試薬および遊離結合剤が、独立して、ストレプトアビジン結合分子;ビオチン;D-ビオチン;ビオチン類似体;ストレプトアビジン、または野生型ストレプトアビジンの位置44～47に対応する配列位置にアミノ酸配列Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ もしくはIle ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ を有するストレプトアビジン類似体に特異的に結合するビオチン類似体からなる群からの分子を含むか、または
競合試薬および遊離結合剤が、独立して、任意でEDTAまたはEGTAである、金属キレート剤を含む、
本発明1116～1121のいずれかの方法。
[本発明1123]
競合試薬および遊離結合剤が、独立して、D-ビオチン、任意で1mMのD-ビオチンを含む、本発明1116～1122のいずれかの方法。
[本発明1124]
細胞を洗浄する工程をさらに含み、任意で、該洗浄工程が組成物中の刺激試薬および/または1つもしくは複数の刺激物質を低減または除去する、本発明1081～1123のいずれかの方法。
[本発明1125]
洗浄する工程が採取前に実行される、本発明1124の方法。
[本発明1126]
T細胞が、抗原特異的T細胞もしくはその集団、Tヘルパー細胞もしくはその集団、細胞傷害性T細胞もしくはその集団、メモリーT細胞もしくはその集団、または制御性T細胞もしくはその集団を含む、本発明1001～1125のいずれかの方法。
[本発明1127]
T細胞がCD3＋T細胞を含むか、CD4＋および/またはCD8＋T細胞を含む、本発明1001～1126のいずれかの方法。
[本発明1128]
前記組成物の刺激されたT細胞からT細胞サブセットを選択する工程を含み、その後に本発明1081～1093のいずれかの導入が行われ、選択された該T細胞サブセットに組換え核酸分子が導入される、本発明1001～1127のいずれかの方法。
[本発明1129]
形質導入細胞を含む組成物からT細胞サブセットを選択する工程を含み、その後に本発明1094～1099のいずれかのインキュベーションが行われ、選択された該T細胞サブセットがウイルス組込みのための条件下でインキュベートされる、本発明1001～1128のいずれかの方法。
[本発明1130]
操作された細胞を含む組成物からT細胞サブセットを選択する工程を含み、その後に本発明1100～1101のいずれかの培養工程が行われ、選択された該T細胞サブセットが、T細胞を拡大培養するための条件下で培養される、本発明1001～1129のいずれかの方法。
[本発明1131]
操作された細胞を含む組成物からT細胞サブセットを選択する工程を含み、その後に本発明1102～1109のいずれかの採取が行われ、選択された該T細胞サブセットが、操作されたT細胞のアウトプット集団を作製するために採取される、本発明1001～1130のいずれかの方法。
[本発明1132]
T細胞サブセットがナイーブ様T細胞であるか、またはナイーブ様T細胞上に発現するマーカーについて表面陽性であるT細胞がCCR7＋CD45RA＋、CD27＋CCR7＋もしくはCD62L-CCR7＋である、本発明1129～1131のいずれかの方法。
[本発明1133]
ナイーブ様T細胞がCD27＋CCR7＋T細胞を含む、本発明1129または本発明1132の方法。
[本発明1134]
ナイーブ様T細胞がCCR7＋CD45RA＋T細胞を含む、本発明1129または本発明1132の方法。
[本発明1135]
T細胞サブセットが、組換えタンパク質、任意でキメラ抗原受容体を発現する、本発明1129～1134のいずれかの方法。
[本発明1136]
T細胞サブセットを選択する工程がアフィニティーカラムクロマトグラフィーによって実行される、本発明1129～1135のいずれかの方法。
[本発明1137]
採取された細胞を、冷凍保存用および/または対象への投与用に、任意で薬学的に許容される賦形剤の存在下で、製剤化する工程をさらに含む、本発明1102～1108、1125および1129～1136のいずれかの方法。
[本発明1138]
採取された細胞が凍結保護物質の存在下で製剤化される、本発明1102～1108、1125および1129～1137のいずれかの方法。
[本発明1139]
固定相がクロマトグラフィーマトリックスであるか、またはそれを含む、本発明1001～1138のいずれかの方法。
[本発明1140]
固定相が、固定相1mLあたりT細胞約7500万～約1億2500万個の結合容量、任意で静的結合容量または動的結合容量を有する、本発明1001～1139のいずれかの方法。
[本発明1141]
（a）固定相が約20mLであり、および/または
（b）固定相が細胞20億±5億個の結合容量を有する、
本発明1001～1140のいずれかの方法。
[本発明1142]
2つの固定相を含む、本発明1001～1141のいずれかの方法。
[本発明1143]
2つの固定相が並列に配置される、本発明1142の方法。
[本発明1144]
2つの固定相が逐次的に配置される、本発明1142の方法。
[本発明1145]
（a）T細胞表面上の第1分子および第2分子にそれぞれ特異的に結合することによってT細胞を刺激する能力を有する、第1刺激物質および第2刺激物質と、
（b）T細胞上の選択マーカーに特異的に結合することによって固定相上にT細胞を固定化する能力を有する選択物質を含む、固定相と
を含む、T細胞のオンカラム刺激のための製造物品。
[本発明1146]
固定相が第1刺激物質および第2刺激物質をさらに含む、本発明1145の製造物品。
[本発明1147]
第1刺激物質、第2刺激物質および選択物質が、選択試薬を介して間接的に固定相に結合されている、本発明1145または本発明1146の製造物品。
[本発明1148]
刺激試薬をさらに含み、第1刺激物質および第2刺激物質が、可逆的に結合されているかまたは可逆的に結合される能力を有する、本発明1145の製造物品。
[本発明1149]
選択物質が選択試薬を介して間接的に固定相に結合されている、本発明1145の製造物品。
[本発明1150]
固定相が、クロマトグラフィーマトリックスであるかそれを含み、前記製造物品が、該クロマトグラフィーマトリックスの全部または一部を含有する容器をさらに含む、本発明1145～1149のいずれかの製造物品。
[本発明1151]
2つの固定相を含む、本発明1145～1150のいずれかの製造物品。
[本発明1152]
2つの固定相が並列に配置される、本発明1151の製造物品。
[本発明1153]
2つの固定相が逐次的に配置される、本発明1151の製造物品。
[本発明1154]
本発明1145～1153のいずれかの製造物品を含む、装置。
[本発明1155]
装置の1つまたは複数の構成要素に流体的に接続された流体入口および/あるいは装置の1つまたは複数の構成要素に流体的に接続された流体出口をさらに含む、本発明1154の装置。
[本発明1156]
閉鎖システム内または無菌システム内にある、本発明1154または1155のいずれかの装置。
[本発明1157]
自動で実行されてもよい本発明1001～1144のいずれかの方法において使用するための、本発明1154～1156のいずれかの装置または本発明1145～1153のいずれかの物品。
Apparatus and/or articles of manufacture are provided for use in any of the methods provided herein (including aspects thereof) that may be performed automatically.
[The present invention 1001]
A method for on-column stimulation of T cells, comprising the steps of:
adding an oligomeric stimulatory reagent capable of delivering a stimulatory signal in a T cell to a stationary phase comprising a plurality of immobilized T cells, thereby initiating incubation of the stimulatory reagent with the one or more T cells,
the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells or a subset thereof;
the oligomeric stimulation reagent comprises one or more stimulatory agents including (i) a first stimulatory agent that is an anti-CD3 antibody and (ii) a second stimulatory agent that is an anti-CD28 antibody;
process; and
Harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of the start of incubation, thereby producing a composition comprising stimulated T cells.
[The present invention 1002]
A method for on-column stimulation of T cells, comprising the steps of:
(a) incubating a plurality of T cells immobilized on a stationary phase with one or more stimulatory agents to deliver a stimulatory signal in one or more T cells of the plurality of T cells, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker on a surface of the one or more T cells, and specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes the one or more T cells on the stationary phase; and
(b) harvesting the one or more T cells from the stationary phase by gravity flow within 24 hours of the start of incubation, thereby producing a composition comprising stimulated T cells.
[The present invention 1003]
The method of the present invention 1002, wherein the immobilized phase comprises at least one of one or more stimulatory substances capable of delivering a stimulatory signal in one or more T cells or is immobilized together with at least one of the one or more stimulatory substances.
[The present invention 1004]
The method of the present invention 1002, wherein the one or more stimulating substances are a first stimulating substance and a second stimulating substance, and prior to incubation, a stimulating reagent including a second stimulating substance capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating substance is added to the stationary phase.
[The present invention 1005]
The method of claim 1002, further comprising the step of adding a stimulating reagent comprising at least one of the one or more stimulating substances to the stationary phase prior to incubating.
[The present invention 1006]
The method of the present invention 1005, wherein at least one stimulating agent is a first stimulating agent, and the one or more stimulating agents further comprise a second stimulating agent capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating agent.
[The present invention 1007]
Any of the methods of 1002-1006, wherein at least one of the one or more stimulatory substances, optionally the first stimulatory substance, is capable of delivering a stimulatory signal, the stimulatory signal being a stimulatory signal via the TCR/CD3 complex in the T cell, a CD3-containing complex in the T cell, and/or an ITAM-containing molecule in the T cell.
[The present invention 1008]
The method of any of claims 1004, 1006 and 1007, wherein the second stimulatory agent has the ability to specifically bind to one or more costimulatory molecules on T cells.
[The present invention 1009]
A method for on-column stimulation of T cells, comprising the steps of:
(a) adding a sample comprising a plurality of T cells to a stationary phase, the stationary phase comprising a selection agent that binds to a selection marker on a surface of one or more of the plurality of T cells, thereby immobilizing the one or more of the plurality of T cells on the stationary phase;
(b) adding a stimulatory reagent to the stationary phase, the stimulatory reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulatory reagent with the one or more T cells; and
(c) harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of the start of incubation, thereby producing a composition comprising stimulated T cells.
[The present invention 1010]
A method for on-column stimulation of T cells, comprising the steps of:
(1) mixing (a) a sample comprising a plurality of T cells with (b) a stationary phase comprising a selection agent capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, wherein specific binding of the selection agent to the selection marker immobilizes the one or more of the plurality of T cells to the stationary phase;
(2) adding a stimulatory reagent to the stationary phase, the stimulatory reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in the T cells, thereby initiating incubation of the stimulatory reagent with the one or more T cells; and
(3) harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of the start of incubation, thereby producing a composition comprising stimulated T cells.
[The present invention 1011]
A method for on-column stimulation of T cells, comprising the steps of:
adding an oligomeric stimulating reagent to a stationary phase comprising the plurality of immobilized T cells, thereby initiating incubation of the stimulating reagent with one or more T cells of the plurality of immobilized T cells;
the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells, and specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes the one or more T cells to the stationary phase;
the oligomeric stimulating reagent comprises (i) a plurality of streptavidin or streptavidin mutein molecules and (ii) one or more stimulatory substances capable of delivering a stimulatory signal in one or more T cells, the size of the oligomeric stimulating reagent comprising (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5×10 ⁶ g/mol, and/or (iii) at least 100 streptavidin or streptavidin mutein tetramers per oligomeric stimulating reagent;
Process.
[The present invention 1012]
The method of claim 1011, further comprising the step of harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of initiating incubation, thereby producing a composition comprising stimulated T cells.
[The present invention 1013]
The method of any of claims 1001-1010 or 1012, wherein harvesting one or more of the plurality of T cells from the stationary phase occurs within about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 hours from the start of incubation.
[The present invention 1014]
Harvesting one or more of the plurality of T cells from the stationary phase may be performed within about 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 19-24, 20-24, 21-24, 22-24, 23-24, 24-24, 25-24, 26-24, 27-24, 28-24, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 42-44, 43-46, 44-48, 45-49, 46-50, 47-51, 48-52, 49-53, 50-54, 51-55, 52-56, 53-57, 54-58, 55-59, 56-60, 57-61, 58-62, 59-70, 61-62, 62-63, 63-64, 64-65, 65-70, 66-67, 67-71, 68-72, 69-80, 70-81, 71-82, 72-83, 73-84, 74-85, 75-86, 76-87, 77-88, 78-90, 79-100 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-30, 2-32, 2-34, 2-36, 2-38, 2-40, 2-42, 2-46, 2-50, 2-44, 2-52, 2-46, 2-54, 2-48, 2-56, 2-48, 2-58, 2-48, 2-56 ...
[The present invention 1015]
The method of any of claims 1001-1010 or 1012-1014, wherein harvesting one or more of the plurality of T cells from the stationary phase occurs within about 12, 10, 8, 6, 4 or 2 hours from the start of incubation.
[The present invention 1016]
The method of any of claims 1001-1010 or 1012-1015, wherein harvesting one or more of the plurality of T cells from the stationary phase is performed within 5 hours of initiating incubation.
[The present invention 1017]
The method of any of claims 1001-1010 or 1012-1016, wherein harvesting one or more of the plurality of T cells from the stationary phase occurs within or about 4.5 hours from the start of incubation.
[The present invention 1018]
The method of any of claims 1001-1010 or 1012-1017, wherein harvesting one or more of the plurality of T cells from the stationary phase is performed within 4 hours of initiating incubation.
[The present invention 1019]
Any of the methods of inventions 1001, 1009, 1010 and 1013-1017, wherein the initiation of incubation with the stimulating reagent is carried out within or within 10 minutes, 20 minutes or within or within about 20 minutes, 30 minutes or within or within about 30 minutes, 45 minutes or within or within about 45 minutes, 60 minutes or within or within about 60 minutes, 90 minutes or within or within about 90 minutes, or 120 minutes or within or within about 120 minutes after the sample comprising the plurality of T cells is added to or mixed with the stationary phase.
[The present invention 1020]
The method of any of claims 1001, 1009, 1010 and 1013-1019, wherein the initiation of incubation with the stimulating reagent is performed within 60 minutes or within about 60 minutes after adding the sample containing the plurality of T cells to or mixing with the stationary phase.
[The present invention 1021]
Any of the methods of claims 1009 to 1020, wherein at least one of the one or more stimulatory substances is capable of delivering a stimulatory signal, the stimulatory signal being a stimulatory signal via the TCR/CD3 complex in the T cell, a CD3-containing complex in the T cell, and/or an ITAM-containing molecule in the T cell.
[The present invention 1022]
The method of the present invention 1021, wherein at least one stimulating substance is a first stimulating substance and the stimulating reagent further comprises one or more second stimulating substances capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating substance.
[The present invention 1023]
The method of any of claims 1004, 1006, 1007 and 1022, wherein the second stimulatory agent has the ability to specifically bind to one or more costimulatory molecules on T cells.
[The present invention 1024]
The method of claim 1008 or claim 1023, wherein the costimulatory molecule is selected from among CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM.
[The present invention 1025]
The method of claim 1008, claim 1023 or claim 1024, wherein the second stimulatory agent has the ability to specifically bind to CD28 and/or the costimulatory molecule is CD28.
[The present invention 1026]
The method of any of claims 1014, 1006, 1007, 1008 and 1020-1023, wherein the first stimulatory agent specifically binds to CD3 and the second stimulatory agent specifically binds to CD28.
[The present invention 1027]
the one or more stimulatory substances are or include, independently, substances selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof;
Any of the methods of the present invention 1001 to 1026.
[The present invention 1028]
the first stimulatory agent and the second stimulatory agent are, or comprise, independently, a substance selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof;
Any of the methods of the present invention 1004, 1006, 1007, 1008 or 1013-1020, 1022-1264.
[The present invention 1029]
The method of any of claims 1001-1027, wherein the one or more stimulatory agents comprises a monovalent antibody fragment.
[The present invention 1030]
The method of any of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026 and 1028, wherein the first stimulatory agent and the second stimulatory agent independently comprise a monovalent antibody fragment.
[The present invention 1031]
The method of any of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026, 1028 and 1030, wherein the first stimulatory agent comprises a monovalent antibody fragment that binds to CD3 and the second stimulatory agent comprises a monovalent antibody fragment that binds to CD28.
[The present invention 1032]
The method of any of claims 1027 to 1031, wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment and a single chain Fv fragment (scFv).
[The present invention 1033]
The method of any one of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026, 1028 and 1030-1032, wherein the first stimulatory agent is an anti-CD3 Fab and the second stimulatory agent is an anti-CD28 Fab.
[The present invention 1034]
A method for on-column stimulation of T cells, comprising the steps of:
adding an oligomeric stimulatory reagent capable of delivering a stimulatory signal in a T cell to a stationary phase comprising a plurality of immobilized T cells, thereby initiating incubation of the stimulatory reagent with the one or more T cells,
the stationary phase comprises a selection agent capable of specifically binding to a selection marker on the surface of one or more T cells or a subset thereof, the specific binding of the selection agent to the selection marker expressed by the one or more T cells or a subset thereof immobilizes the at least a plurality of T cells to the stationary phase, the selection agent being a Fab fragment capable of specifically binding to a selection marker selected from the group consisting of CD3, CD4 and CD8;
the oligomeric stimulatory reagent comprises (i) a plurality of streptavidin mutein molecules, (ii) a first stimulatory agent which is a Fab fragment capable of specifically binding to CD3 and capable of delivering a stimulatory signal in one or more T cells, and (iii) a second stimulatory agent which is a Fab fragment capable of specifically binding to CD28 and capable of enhancing, attenuating or modifying the stimulatory signal, the size of the oligomeric stimulatory reagent comprising (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5×10 ⁶ g/mol, and/or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulatory reagent;
Process; and
Harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours of the start of incubation, thereby producing a composition comprising stimulated T cells.
[The present invention 1035]
The method of any of claims 1001-1034, wherein the T cells are derived from a sample that is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product.
[The present invention 1036]
The method of any of claims 1035, wherein the sample is an apheresis product or a leukapheresis product.
[The present invention 1037]
Any of the methods of the present invention 1036, wherein the apheresis product or leukapheresis product has been previously cryofrozen.
[The present invention 1038]
The method of any of claims 1002-1027 and 1029, wherein incubating with the one or more stimuli releases one or more of the plurality of immobilized T cells from the stationary phase.
[The present invention 1039]
The method of any of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026, 1028, 1030-1038, wherein incubating with the first stimulatory agent and the second stimulatory agent releases one or more of the plurality of immobilized T cells from the stationary phase.
[The present invention 1040]
a biotin analogue in which one or more stimuli reversibly bind to biotin, streptavidin or avidin;

The method of any of claims 1001 to 1027, 1029 and 1035 to 1028, further comprising a streptavidin-binding peptide selected from the group consisting of a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.
[The present invention 1041]
One or more stimulants

The method of any one of claims 1001 to 1027, 1029 and 1035 to 1040, further comprising a streptavidin binding peptide selected from the group consisting of:
[The present invention 1042]
One or more stimuli are

The method of any of claims 1001-1027, 1029 and 1035-1041, further comprising a streptavidin binding peptide having the formula:
[The present invention 1043]
the first stimulatory agent and the second stimulatory agent are, independently, biotin analogs that reversibly bind to biotin, streptavidin, or avidin;

The method of any one of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026, 1028, and 1030-1039, further comprising a streptavidin-binding peptide selected from the group consisting of a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.
[The present invention 1044]
Each of the first stimulus substance and the second stimulus substance is

The method of any one of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026, 1028, 1030-1039 and 1043, further comprising a streptavidin binding peptide selected from the group consisting of:
[The present invention 1045]
Each of the first and second stimulus substances has the sequence

The method of any of claims 1001, 1004, 1006, 1007, 1008, 1013-1020, 1022-1026, 1028, 1030-1039, 1043 and 1044, further comprising a streptavidin binding peptide having the formula:
[The present invention 1046]
The method of any of claims 1001 to 1045, wherein the selected agent is or comprises an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof.
[The present invention 1047]
the selection marker is a T cell coreceptor;
the selectable marker is or comprises a member of the T cell antigen receptor complex;
the selection marker is or comprises the CD3 chain,
the selection marker is or comprises the CD3 zeta chain,
the selection marker is or comprises CD8;
the selection marker is or comprises CD4,
the selection marker is or comprises CD45RA;
the selection marker is or comprises CD27,
the selection marker is or comprises CD28, and/or
The selection marker is or comprises CCR7,
Any of the methods of the present invention 1001 to 1046.
[The present invention 1048]
The method of any of claims 1001 to 1047, wherein the selectable marker is selected from the group consisting of CD3, CD4 and CD8.
[The present invention 1049]
The method of any of claims 1001 to 1048, wherein the selectable marker is CD3.
[The present invention 1050]
the selective agent is a biotin analogue that reversibly binds to biotin, streptavidin or avidin;

Any of the methods of claims 1001 to 1049, further comprising a streptavidin-binding peptide selected from the group consisting of a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.
[The present invention 1051]
the selective agent is a biotin analogue that reversibly binds to biotin, streptavidin or avidin;

The method of any one of claims 1001 to 1050, further comprising a streptavidin binding peptide selected from the group consisting of:
[The present invention 1052]
The selected substance has the sequence

The method of any of claims 1001 to 1051, further comprising a streptavidin binding peptide having the formula:
[The present invention 1053]
The method of any of claims 1001 to 1052, wherein the specific binding between the selection substance and the selection marker does not induce a signal to the T cell, or does not induce any stimulatory, activating or proliferative signal to the T cell.
[The present invention 1054]
The method of any of claims 1001 to 1053, wherein the selection agent comprises a monovalent antibody fragment that binds to CD3, CD8 or CD4.
[The present invention 1055]
The method of any of claims 1001 to 1054, wherein the selection agent is an anti-CD3 Fab, an anti-CD8 Fab or an anti-CD4 Fab.
[The present invention 1056]
The method of any of claims 1001 to 1055, wherein the selecting agent is an anti-CD3 Fab.
[The present invention 1057]
Any of the methods of claims 1004 to 1010, 1013 to 1033 and 1035 to 1056, wherein the stimulation reagent is an oligomeric stimulation reagent comprising a plurality of streptavidin molecules or streptavidin mutein molecules, and the size of the oligomeric particle reagent comprises (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5 x 10 ⁶ g/mol and/or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulation reagent.
[The present invention 1058]
The method of the present invention 1001, wherein the oligomeric stimulation reagent comprises a plurality of streptavidin molecules or streptavidin mutein molecules, and the size of the oligomeric particle reagent is (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5 x 10 ⁶ g/mol, and/or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulation reagent.
[The present invention 1059]
The method of any one of claims 1001, 1011-1058, wherein the oligomeric stimulating reagent is soluble.
[The present invention 1060]
the oligomeric stimulation reagent is not, and is not bound to or associated with, a solid support, stationary phase, bead, microparticle, magnetic particle, and/or matrix; and/or
the reagent is flexible, does not contain a metallic or magnetic core, is composed entirely or primarily of organic polymers, and/or is not rigid;
Any of the methods of the present invention 1001, 1011 to 1059.
[The present invention 1061]
The method of any of claims 1011-1060, wherein the streptavidin or streptavidin mutein molecule reversibly binds or has the ability to reversibly bind to biotin, a biotin analogue or a streptavidin binding peptide.
[The present invention 1062]
the streptavidin mutein comprises the amino acid sequence ^Ile44 - ^Gly45 - ^Ala46 - ^Arg47 at sequence positions corresponding to positions 44 to 47 in streptavidin relative to the positions in the sequence of amino acids shown in SEQ ID NO:1; or
The streptavidin mutein comprises the amino acid sequence Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44 to 47 in streptavidin based on the amino acid sequence shown in SEQ ID NO:1,
Any of the methods of the present invention 1011 to 1061.
[The present invention 1063]
Streptavidin-binding peptides

3. The method of claim 1061 or 1062, wherein the method is selected from the group consisting of:
[The present invention 1064]
The oligomer stimulating agent is
Radius greater than 60nm, greater than 70nm, greater than 80nm or greater than 90nm
The method of any one of claims 1001 and 1011 to 1063, comprising:
[The present invention 1065]
The oligomer stimulating agent is
a radius between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm, inclusive; or
Radius of 90nm±15nm or 95nm±20～25nm
The method of any one of claims 1001 and 1011 to 1064, comprising:
[The present invention 1066]
Any of the methods of any of claims 1164 to 1167, wherein the radius is a hydrodynamic radius.
[The present invention 1067]
The oligomer stimulating agent is
at least 5×10 ⁷ g/mol or at least 1×10 ⁸ g/mol, and/or
5×10 ⁷ g/mol to 5×10 ⁸ g/mol, 1×10 ⁸ g/mol to 5×10 ⁸ g/mol, or 1×10 ⁸ g/mol to 2×10 ⁸ g/mol
The method of any of claims 1001 and 1011 to 1066, comprising the step of:
[The present invention 1068]
The oligomer stimulating agent is
at least 500 streptavidin tetramers or streptavidin mutein tetramers, at least 1,000 streptavidin tetramers or streptavidin mutein tetramers, at least 1,500 streptavidin tetramers or streptavidin mutein tetramers, or at least 2,000 streptavidin tetramers or streptavidin mutein tetramers, and/or
1,000 to 20,000 streptavidin tetramers or streptavidin mutein tetramers, 1,000 to 10,000 streptavidin tetramers or streptavidin mutein tetramers, or 2,000 to 5,000 streptavidin tetramers or streptavidin mutein tetramers
The method of any one of claims 1001 and 1011 to 1067,
[The present invention 1069]
The method of any of claims 1001 and 1011-1068, wherein the oligomeric stimulating reagent is added to the stationary phase at a concentration of about 1 to about 2 μg/million cells.
[The present invention 1070]
The method of any of claims 1001-1069, wherein the selection agent is directly or indirectly bound to the stationary phase.
[The present invention 1071]
The method of any of claims 1001-1070, wherein the selection substance is indirectly bound to the stationary phase via a selection reagent to which the selection substance reversibly binds.
[The present invention 1072]
The method of claim 1070 or 1071, wherein the selection reagent is or comprises streptavidin; avidin; a mutein of streptavidin which binds reversibly to biotin, a biotin analogue or a biologically active fragment thereof; an avidin or a mutein of streptavidin which binds reversibly to a streptavidin-binding peptide; a reagent comprising at least two chelating groups K, said at least two chelating groups being capable of binding to a transition metal ion; a substance capable of binding to an oligohistidine affinity tag; a substance capable of binding to glutathione-S-transferase; calmodulin or an analogue thereof; a substance capable of binding to calmodulin-binding peptide (CBP); a substance capable of binding to a FLAG peptide; a substance capable of binding to an HA tag; a substance capable of binding to maltose binding protein (MBP); a substance capable of binding to an HSV epitope; a substance capable of binding to a myc epitope; or a substance capable of binding to a biotinylated carrier protein.
[The present invention 1073]
the selection agent is or comprises a streptavidin or avidin mutein that reversibly binds biotin or a biologically active fragment;
the stimulating agent is or comprises a streptavidin or avidin mutein that reversibly binds to a biotin analog or biologically active fragment; and/or
The stimulating reagent is or comprises a streptavidin or avidin mutein that reversibly binds to a streptavidin binding peptide;
Any of the methods of 1070 to 1072 of the present invention.
[The present invention 1074]
1074. The method of claim 1072 or 1073, wherein the streptavidin molecule or the streptavidin mutein molecule reversibly binds or has the ability to reversibly bind to biotin, a biotin analogue or a streptavidin binding peptide.
[The present invention 1075]
the streptavidin mutein comprises the amino acid sequence ^Ile44 - ^Gly45 - ^Ala46 - ^Arg47 at sequence positions corresponding to positions 44 to 47 in streptavidin relative to the positions in the sequence of amino acids shown in SEQ ID NO:1; or
The streptavidin mutein comprises the amino acid sequence Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44 to 47 in streptavidin based on the amino acid sequence shown in SEQ ID NO:1,
Any of the methods of claims 1072 to 1074.
[The present invention 1076]
Streptavidin-binding peptides

Any of the methods of claims 1072 to 1075, selected from the group consisting of:
[The present invention 1077]
The streptavidin binding peptide has the sequence

Any of the methods of claims 1072 to 1076, comprising the steps of:
[The present invention 1078]
Any of the methods of claims 1001-1010 or 1012-1077, wherein collecting by gravity flow comprises adding medium to the stationary phase that does not contain a competitor or a free binding agent for eluting the T cells from the stationary phase.
[The present invention 1079]
The method of any of claims 1001-1010 and 1012-1078, wherein the composition comprising the stimulated T cells does not comprise a competitor or a free binder.
[The present invention 1080]
1079. The method of any one of claims 1078 to 1079, wherein the competitor or free binder is or comprises biotin or a biotin analogue, optionally wherein the biotin analogue is D-biotin.
[The present invention 1081]
The method of any of claims 1001-1078, further comprising the step of introducing a recombinant nucleic acid molecule encoding a recombinant protein into the stimulated T cells of said composition, thereby producing a composition comprising engineered T cells, optionally a composition comprising transduced T cells.
[The present invention 1082]
The method of claim 1081, wherein the recombinant protein is an antigen receptor.
[The present invention 1083]
The method of claim 1081 or 1082, wherein the recombinant protein is a chimeric antigen receptor.
[The present invention 1084]
The method of the present invention, wherein the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM.
[The present invention 1085]
The method of claim 1084, wherein the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.
[The present invention 1086]
The method of claim 1084 or 1085, further comprising a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
[The present invention 1087]
The method of claim 1086, wherein the transmembrane domain comprises the transmembrane portion of CD28.
[The present invention 1088]
The method of any of claims 1084 to 1087, wherein the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule.
[The present invention 1089]
The method of claim 1088, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.
[The present invention 1090]
The method of any of claims 1081 to 1089, wherein said nucleic acid further comprises a promoter operably linked to the nucleic acid encoding the recombinant antigen receptor.
[The present invention 1091]
The method of any of claims 1081 to 1090, wherein the introduction of the recombinant nucleic acid is achieved by transduction with a viral particle.
[The present invention 1092]
The method of claim 1091, wherein the viral particle is a retroviral vector particle.
[The present invention 1093]
The method of claim 1091 or 1092, wherein the viral particle is a lentiviral vector particle.
[The present invention 1094]
The method of any of claims 1081 to 1093, further comprising the step of incubating the composition comprising the transduced cells under conditions for viral integration, optionally at a temperature of or about 37°±2°C.
[The present invention 1095]
The method of claim 1094, wherein the step of incubating the composition comprising the transduced cells is carried out for up to 96 hours after transduction.
[The present invention 1096]
The method of claim 1094, wherein the step of incubating the composition comprising the transduced cells is carried out for up to 72 hours after transduction.
[The present invention 1097]
The method of claim 1094, wherein the step of incubating the composition comprising the transduced cells is carried out for up to 48 hours after transduction.
[The present invention 1098]
The method of claim 1094, wherein the step of incubating the composition comprising the transduced cells is carried out for up to 24 hours after transfection.
[This invention 1099]
The method of any of claims 1094 to 1098, wherein the step of incubating the composition comprising the transduced cells is carried out for at least 18 hours after transfection.
[The present invention 1100]
The method of any of claims 1081-1099, further comprising culturing the composition comprising the engineered cells, optionally the transduced cells, under conditions for expanding the T cells.
[The present invention 1101]
The method of claim 1100, wherein the culturing step is carried out for a period of time of 14 days or less, 12 days or less, 10 days or less, 8 days or less, 6 days or less, or 5 days or less.
[The present invention 1102]
The method of any of claims 1081 to 1101, further comprising the step of harvesting the engineered T cells, thereby generating an output population of engineered T cells.
[The present invention 1103]
The method of any of claims 1081-1099, further comprising harvesting the engineered T cells at a time point between 48 and 120 hours, inclusive, after initiation of exposure to the stimulating agent.
[The present invention 1104]
The method of any of claims 1081-1099 and 1103, wherein the sampling is performed within 120 hours after initiating exposure to the irritant.
[The present invention 1105]
The method of any of claims 1081-1099 and 1103, wherein the sampling is performed within 96 hours after initiating exposure to the irritant.
[The present invention 1106]
The method of any of claims 1081-1099 and 1105, wherein the sampling is performed within 72 hours after initiating exposure to the irritant.
[The present invention 1107]
The method of any of claims 1081-1099 and 1106, wherein the sampling is performed within 48 hours after initiating exposure to the irritant.
[The present invention 1108]
The method of any of claims 1102 to 1107, wherein the percentage of naive-like cells is greater than or greater than about 60% of the total T cells in the population, the total CD4+ T cells in the population, or the total CD8+ T cells or recombinant protein-expressing cells thereof in the population at the time of harvest.
[The present invention 1109]
The method of claim 1108, wherein the naive-like T cells comprise CD27+CCR7+ cells.
[The present invention 1110]
The method of any of claims 1081 to 1093, wherein the introducing is carried out in serum-free medium.
[The present invention 1111]
The method of any of claims 1094 to 1099, wherein the incubating step is carried out in serum-free medium.
[The present invention 1112]
The method of claim 1100 or 1101, wherein the culturing step is carried out in a serum-free medium.
[The present invention 1113]
Serum-free medium,
In the basal medium, 0.5 mM to 5 mM dipeptide-type L-glutamine,
0.5mM to 5mM L-glutamine, and
Optionally, at least one protein
1113. The method of any of claims 1110 to 1112, wherein the medium is serum-free.
[The present invention 1114]
The method of any of claims 1110 to 1113, wherein the serum-free medium comprises a recombinant cytokine selected from among IL-2, IL-15 and IL-7, optionally recombinant human IL-2, recombinant human IL-15 and/or recombinant human IL-7.
[The present invention 1115]
The method of any of claims 1110 to 1114, wherein the serum-free medium does not contain recombinant cytokines selected from among IL-2, IL-15 and IL-7, optionally recombinant human IL-2, recombinant human IL-15 and/or recombinant human IL-7.
[The present invention 1116]
The method of any of claims 1081-1093, further comprising the step of adding a competitor or free binder to the composition comprising the engineered cells, optionally a composition comprising the transduced T cells, optionally under conditions to dissociate one or more stimulatory agents from the oligomeric stimulatory reagent in the composition.
[The present invention 1117]
Any of the methods of 1094-1099, further comprising adding a competitor or free binder to a composition comprising the incubated T cells, optionally under conditions to dissociate one or more stimulatory agents from the oligomeric stimulatory reagent in the composition.
[The present invention 1118]
Any of the methods of 1100-1101, further comprising the step of adding a competitor or free binder to the composition comprising the cultured T cells, optionally under conditions to dissociate one or more stimulatory agents from the oligomeric stimulatory reagent in the composition.
[The present invention 1119]
The method of any one of claims 1116 to 1118, wherein the step of adding a competitor or free binder is carried out prior to harvesting.
[The present invention 1120]
Any of the methods of claims 1116 to 1119, wherein the competitor or free binding agent is not harmful to the T cells and/or addition of the agent does not reduce the percentage of surviving T cells to less than 90%, less than 80%, less than 70%, less than 60% or less than 50% compared to incubation of T cells under equivalent or identical conditions without the use of either the competitor or the free binding agent.
[The present invention 1121]
The method of any of claims 1116 to 1120, wherein said dissociation terminates or reduces a stimulatory signal in the T cell.
[The present invention 1122]
the competing reagent and the free binder independently comprise molecules from the group consisting of streptavidin binding molecules; biotin; D-biotin; a biotin analogue; a biotin analogue that specifically binds to streptavidin or a streptavidin analogue having the amino acid sequence Val44 ^{- Thr45} - ^Ala46 - ^Arg47 or Ile44 ^- Gly45 - ^Ala46 - ^Arg47 at sequence positions corresponding to positions ^44-47 of wild type streptavidin; or
the competitive reagent and the free binding agent independently comprise a metal chelator, optionally EDTA or EGTA;
Any of the methods of the present invention 1116 to 1121.
[The present invention 1123]
The method of any of claims 1116-1122, wherein the competing reagent and the free binding agent independently comprise D-biotin, optionally at 1 mM D-biotin.
[The present invention 1124]
The method of any of claims 1081 to 1123, further comprising a step of washing the cells, optionally wherein said washing step reduces or removes the stimulating reagent and/or one or more stimulating substances in the composition.
[The present invention 1125]
The method of any one of claims 1124 to 1126, wherein a washing step is performed prior to harvesting.
[The present invention 1126]
The method of any of claims 1001-1125, wherein the T cells comprise antigen-specific T cells or a population thereof, T helper cells or a population thereof, cytotoxic T cells or a population thereof, memory T cells or a population thereof, or regulatory T cells or a population thereof.
[The present invention 1127]
The method of any of claims 1001-1126, wherein the T cells comprise CD3+ T cells, or comprise CD4+ and/or CD8+ T cells.
[The present invention 1128]
Any of the methods of claims 1001 to 1127, comprising a step of selecting a T cell subset from the stimulated T cells of said composition, followed by introduction of any of claims 1081 to 1093, and introducing a recombinant nucleic acid molecule into the selected T cell subset.
[The present invention 1129]
The method of any one of claims 1001 to 1128, further comprising the step of selecting a T cell subset from a composition comprising the transduced cells, followed by the incubation of any one of claims 1094 to 1099, wherein the selected T cell subset is incubated under conditions for viral integration.
[The present invention 1130]
The method of any one of claims 1001 to 1129, comprising a step of selecting a T cell subset from a composition comprising the engineered cells, followed by a culturing step of any one of claims 1100 to 1101, wherein the selected T cell subset is cultured under conditions for expanding the T cells.
[The present invention 1131]
The method of any of claims 1001-1130, comprising the step of selecting a T cell subset from a composition comprising the engineered cells, followed by harvesting according to any of claims 1102-1109, and harvesting said selected T cell subset to generate an output population of engineered T cells.
[The present invention 1132]
The method of any of claims 1129 to 1131, wherein the T cell subset is naive-like T cells or the T cells that are surface positive for a marker expressed on naive-like T cells are CCR7+CD45RA+, CD27+CCR7+ or CD62L-CCR7+.
[The present invention 1133]
The method of claim 1129 or 1132, wherein the naive-like T cells comprise CD27+CCR7+ T cells.
[The present invention 1134]
The method of claim 1129 or 1132, wherein the naive-like T cells comprise CCR7+CD45RA+ T cells.
[This invention 1135]
The method of any of claims 1129 to 1134, wherein the T cell subset expresses a recombinant protein, optionally a chimeric antigen receptor.
[The present invention 1136]
The method of any of claims 1129 to 1135, wherein the step of selecting the T cell subset is carried out by affinity column chromatography.
[This invention 1137]
The method of any of claims 1102-1108, 1125 and 1129-1136, further comprising formulating the harvested cells for cryopreservation and/or administration to a subject, optionally in the presence of a pharma- ceutically acceptable excipient.
[The present invention 1138]
The method of any of claims 1102-1108, 1125 and 1129-1137, wherein the harvested cells are formulated in the presence of a cryoprotectant.
[The present invention 1139]
The method of any one of claims 1001 to 1138, wherein the stationary phase is or comprises a chromatographic matrix.
[The present invention 1140]
The method of any of claims 1001-1139, wherein the stationary phase has a binding capacity, optionally a static binding capacity or a dynamic binding capacity, of about 75 million to about 125 million T cells per mL of stationary phase.
[This invention 1141]
(a) the stationary phase is about 20 mL; and/or
(b) the stationary phase has a binding capacity of 2 billion ± 500 million cells;
Any of the methods of the present invention 1001 to 1140.
[This invention 1142]
The method of any of claims 1001 to 1141, comprising two stationary phases.
[This invention 1143]
The method of claim 1142, wherein the two stationary phases are arranged in parallel.
[This invention 1144]
The method of claim 1142, wherein the two stationary phases are arranged sequentially.
[This invention 1145]
(a) a first stimulatory agent and a second stimulatory agent capable of stimulating a T cell by specifically binding to a first molecule and a second molecule, respectively, on the surface of the T cell;
(b) a stationary phase comprising a selection agent capable of immobilizing T cells on the stationary phase by specifically binding to a selection marker on the T cells;
1. An article of manufacture for on-column stimulation of T cells comprising:
[This invention 1146]
1145. The article of manufacture of claim 1145, wherein the stationary phase further comprises a first stimulant and a second stimulant.
[This invention 1147]
The article of manufacture of claim 1145 or claim 1146, wherein the first stimulus agent, the second stimulus agent and the selection agent are indirectly bound to the stationary phase via the selection reagent.
[This invention 1148]
1145. The article of manufacture of claim 1145, further comprising a stimulating reagent, wherein the first stimulating agent and the second stimulating agent are reversibly bound or have the capacity to be reversibly bound.
[This invention 1149]
1145. The article of manufacture of claim 1145, wherein the selection substance is indirectly bound to the stationary phase via the selection reagent.
[The present invention 1150]
The article of manufacture of any one of claims 1145 to 1149, wherein the stationary phase is or comprises a chromatography matrix, and the article of manufacture further comprises a vessel containing all or a portion of the chromatography matrix.
[This invention 1151]
The article of manufacture of any of claims 1145 to 1150, comprising two stationary phases.
[This invention 1152]
1151. The article of manufacture of claim 1151, wherein the two stationary phases are arranged in parallel.
[This invention 1153]
1151. The article of manufacture of claim 1151, wherein the two stationary phases are arranged sequentially.
[This invention 1154]
An apparatus comprising any one of the articles of manufacture of the present invention 1145 to 1153.
[This invention 1155]
The device of the present invention 1154 further comprising a fluid inlet fluidly connected to one or more components of the device and/or a fluid outlet fluidly connected to one or more components of the device.
[This invention 1156]
A device of the present invention 1154 or 1155 in a closed or sterile system.
[This invention 1157]
Any of the apparatuses of the present inventions 1154 to 1156 or any of the articles of the present inventions 1145 to 1153 for use in any of the methods of the present inventions 1001 to 1144, which may be carried out automatically.

FIG. 1 is a schematic diagram of an exemplary embodiment of a process for stimulating and selecting target cells. Here, stimulation is performed by incubation of cells, which is at least partially performed in the presence of a support 6 (depicted here as a stationary phase), on which a component of a selection reagent 1 for cell selection is immobilized (panel A). The selection reagent 1 has a binding site for a selection agent 2, which has the ability to bind to a molecule (selection marker) 4 present on some or all of the target cells. The selection agent 2 is added to the support on which the selection reagent 1 is immobilized under conditions such that the reagent and the agent reversibly bind, e.g., via the binding site, to form an oligomeric complex on which the agent multimerizes (panel B). The selection agent can include two or more agents. Alternatively, a complex of the reversibly bound agent and reagent can be added as a complex to the stationary phase for immobilization. As shown, cells 3, including target cells, are mixed with the stationary phase and the multimerized selection agent complex. This causes the target cells to become reversibly immobilized to support 6 via selection agent 2 and reagent (selection reagent) 1 (panel C). Optionally, unbound cells are removed prior to or after addition of stimulatory agent. A complex containing multimerized stimulatory agent 5 reversibly bound to oligomeric stimulatory reagent 7 is added under conditions such that stimulatory agent 5 specifically binds to a molecule on the target cells, thereby inducing or modulating a signal in the immobilized target cells expressing the marker (panel D). Figures 2A and 2B show the results of WST metabolic assays of T cells from three different donors incubated with anti-CD3/anti-CD28 multimerized on different batches of oligomer reagents. Figure 2A summarizes the WST metabolic activity for all tested batches (pools) compared to a reference batch containing anti-CD3/anti-CD28 multimerized on an oligomer backbone with a mean hydrodynamic radius of 36 nm or 101 nm. Figure 2B shows the average WST metabolic activity in T cells from different donors for each tested batch and the reference reagent. Figure 3 depicts the effect of 24-hour on-column stimulation with anti-CD3/anti-CD28 oligomer stimulating reagent on the surface expression of each molecule when CD3, CD4, and CD8, respectively, are used as selection markers to immobilize cells on the stationary phase of a chromatography column. Surface expression patterns are compared to control conditions without on-column stimulation with anti-CD3/anti-CD28 oligomer stimulating reagent. Cells were isolated from apheresis samples applied to the stationary phase. Figure 4 shows exemplary kinetics of downregulation and re-expression of TCR/CD3 complexes upon on-column stimulation with anti-CD3/anti-CD28 oligomer stimulating reagents when CD3 was used as a selection marker to immobilize cells on the column. Cells were isolated from apheresis samples applied to the stationary phase. CD3/TCR complexes were assessed using antibodies against the alpha-beta TCR chains. Figures 5A-5B show the phenotypic and functional characteristics of cultured T cells spontaneously detached during on-column stimulation with anti-CD3/anti-CD28 oligomer stimulating reagents. Figure 5A shows T cell size and CD3, CD69 and CD25 expression 24 hours and 5 days after on-column stimulation. Figure 5B shows the proliferative potential of spontaneously detached cultured T cells. Cells were isolated from apheresis samples applied to the stationary phase and collected using a washing step. See legend to Figure 5A. Figures 6A-6D depict exemplary effects on mTor signaling and viability and growth kinetics of incubating T cells with anti-CD3/anti-CD28 oligomeric stimulatory reagents in the presence or absence of compound 63. Figure 6A depicts pS6 expression by memory subsets in live CD8+ T cells. Figure 6B depicts mean fluorescence intensity (mfi) of total CD8 T cells with the indicated treatments. Figures 6C-6D depict viability and total T cell numbers, respectively, over time (shown in days, e.g., d1) in culture following the initiation of stimulation ("input"). Figures 7A-7F show exemplary functional and phenotypic characteristics of cryopreserved CAR-T cells generated using a method using incubation with anti-CD3/anti-CD28 oligomer stimulating reagents in the presence or absence of compound 63. Figure 7A shows intracellular expression of caspases upon thawing. Figures 7B and 7D show phenotypic profiles of CD8 and CD4 CAR-T cells, respectively, by subset expression of CD27 and/or CCR7. Figures 7C and 7E show intracellular IL2, IFNg or TNF (left panel) or a combination of IL2 and/or IFNg or TNF (right panel) in CD8 and CD4 CAR-T cells, respectively, stimulated with antigen-bearing targets. Figure 7F shows 12-day expansion and survival (left panel) and total expansion metric calculated by area under the curve (right panel) for CAR-T cells stimulated with anti-CAR beads. Figure 8A depicts CD3+, CD4+, and CD8+ T cell yields following cell selection using the on-column stimulation process or an alternative process described in Example 5. Figures 8B-8C depict the total number of cells (Figure 8B) and percentage of viable cells (Figure 8C) recovered following use of the on-column stimulation process or an alternative process described in Example 5. See legend to Figure 8A. See legend to Figure 8A. Figures 9A-9D depict the percentage of live cells (e.g., purity; Figure 9A), the percentage of live cells expressing an exemplary CAR (Figure 9B), the percentage of live cells expressing CD4 at the time of process selection and at day 8 of the process (Figure 9C), and the distribution (percentage) of T cell phenotypes for each donor at day 5 of culture (from the start of the process to day 8) (Figure 9D) for the on-column stimulation process described in Example 5 or an alternative process. See legend to Figure 9A. See legend to Figure 9A. See legend to Figure 9A. Figure 10 depicts CD19+ HEK cell lysis over time during culture with anti-CD19 CAR T cells engineered using the on-column stimulation process described in Example 5 or an alternative process, and under control conditions. Figures 11A-11C depict antigen-specific CAR T cell production of IFNg (Figure 11A), IL-2 (Figure 11B) and TNF-a (Figure 11C) for CD4 and CD8 T cells engineered using the on-column stimulation process described in Example 5 or an alternative process. See legend to Figure 11A. See legend to Figure 11A. Figures 12A-12C show the CD4:CD8 ratio (Figure 12A), transduction efficiency (CD4 and CD8 cells combined; Figure 12B), and percentage of viable cells (Figure 12C) of engineered T cells generated using the on-column stimulation process or an alternative process described in Example 5. Three production runs are shown for each process. See legend to Figure 12A. See legend to Figure 12A. FIG. 13 depicts tumor size by mean radiance in treatment groups 6 days after mice were injected (i.v.) with a B cell lymphoma cell line (Raji). Figure 14 depicts tumor burden in mice injected with a B cell lymphoma cell line (Raji) over time for each treatment group. The effect of CAR T cell treatment is shown for the on-column stimulation process or the alternative process described in Example 5, and for each of the three production runs (see Figures 12A-12C).

DETAILED DESCRIPTION Provided herein are methods for selecting cells from a sample containing target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells), immobilizing the target cells on a stationary phase of a chromatography column, stimulating the immobilized cells on the stationary phase (also referred to herein as on-column stimulation), and collecting and/or eluting the selected and stimulated cells that spontaneously detach from the stationary phase without using a competitor or free binder to promote detachment. Some of the provided methods involve selecting cells from a sample containing target cells (e.g., T cells, CD3+, CD4+, CD8+ T cells), immobilizing the target cells on a stationary phase of a chromatography column, stimulating the immobilized cells on the stationary phase, and collecting and/or eluting the selected and stimulated cells by gravity flow. In provided embodiments, stimulating the target cells (e.g., CD3+, CD4+, or CD8+ T cells) on the stationary phase of the chromatography column promotes downregulation of the molecule used for cell selection (i.e., selection marker), resulting in spontaneous detachment or release of the cells from the stationary phase. The release or detachment of the cells can occur without any additional steps or additional reagents. In some aspects, the cells can be collected by gravity flow, such as by adding medium or other solution to a chromatography column. In certain embodiments, the added medium or other solution does not contain a competitor or free binding agent to promote the release of the cells from the stationary phase.

In some embodiments, the selected stimulated cells are compositions containing stimulated T cells, in which T cells are selected from a biological sample (e.g., an apheresis sample or a whole blood sample) containing a plurality of T cells. In some embodiments, collection and/or elution of selected stimulated cells that spontaneously detach from the stationary phase is accomplished by gravity flow, e.g., during a washing step. In the methods provided herein, cell selection, stimulation, and collection and/or elution steps are integrated, and no separate steps are required to facilitate detachment of the selected stimulated cells from the stationary phase or purification steps to remove agents used to facilitate detachment (e.g., competitors and/or free binding agents). Thus, the methods reduce the number of processing steps required to generate a composition of selected stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion, subsequent incubation, stimulation and/or selection (e.g., initial selection and/or polishing)), thereby reducing production time, minimizing potential cell stress, and reducing the possibility of contamination.

In certain embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within 24 hours. In certain embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours. In certain embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 6, 5, 4, 3, or 2 hours. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 6 hours. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within or about 5.5 hours. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 5 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 4.5 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 4 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 3 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 3-6 hours or less. In some embodiments, the method generates an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 4-6 hours or less. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 5-6 hours or less. In some embodiments, the methods generate an output composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within about 4-5 hours or less. In some embodiments, the methods provided herein generate a composition of engineered T cells (e.g., a therapeutic cell composition) within 5 days. In some embodiments, the methods provided herein generate a composition of engineered T cells (e.g., a therapeutic cell composition) in 4-5 days or about 4-5 days. In some embodiments, the process provided herein results in a manufacturing process that is 4 or 5 days or about 4 or 5 days in length. In some embodiments, the process provided herein results in a manufacturing process that is about 4-5 days in length. In some embodiments, the process provided herein results in a manufacturing process that is 4 days or about 4 days or 96±6 hours in length.

The methods provided include a method for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., other cells in a sample, and immobilizing the cells on a stationary phase of a chromatography column; stimulating the selected cells immobilized on the stationary phase; and collecting the selected stimulated cells in the absence of processing steps to detach the cells from the stationary phase and to remove agents (e.g., competitors or free binders) used to promote detachment from the output composition of the selected stimulated cells. In a particular embodiment, the methods provided include a method for selecting cells, e.g., CD3+, CD4+, and CD8+ T cells, from other components, e.g., other cells in a sample, and immobilizing the cells on a stationary phase of a chromatography column; stimulating the selected cells immobilized on the stationary phase; and eluting and/or collecting the selected stimulated cells by gravity flow.

In certain aspects, the methods provided herein are an improvement over many existing methods that include one or more additional steps prior to stimulating cells after cell selection (e.g., immunoaffinity-based selection) to generate engineered cells (e.g., T cells), such as for cell therapy. In some embodiments, the one or more additional steps present in the existing methods can include one or more elution steps using competitive or free binding agents to recover or collect the selected cells and/or a step to remove a reagent (e.g., magnetic bead reagent or antibody) used for selection. In some embodiments, such additional steps may lengthen the process for genetically engineering cells for cell therapy and/or may result in manipulation of the cells during the process that may impact the differentiation state, viability or cell number of the cells. In certain aspects, the methods provided generate a population of selected and stimulated cells in a reduced time compared to methods that include separate selection and stimulation steps and require additional steps to detach the cells from the stationary phase and additional steps to remove agents used to promote detachment.

In certain aspects, the method produces an output population of selected stimulated cells (also referred to as an output composition) suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 24 hours of initiating on-column stimulation, also referred to herein as on-column stimulation. In some embodiments, the method produces an output population of selected stimulated cells (e.g., output composition) suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within or about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 hours of initiating on-column stimulation. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within or about 6, 5, 4, 3 or 2 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within or about 3-6 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within or about 4-6 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 5-6 hours or about 5-6 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 4-5 hours or about 4-5 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 6 hours or about 6 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 5.5 hours or about 5.5 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 5 hours or about 5 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 4.5 hours or about 4.5 hours. In some embodiments, the method generates an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 4 hours or about 4 hours. In some embodiments, the method produces an output population of selected and stimulated cells suitable for downstream processing (e.g., genetic manipulation, expansion and/or subsequent incubation, stimulation and/or selection rounds (e.g., polishing)) within 3 hours or about 3 hours.

In some embodiments, the method involves the use of a stimulatory agent capable of delivering a stimulatory signal in the cell by binding to a molecule on the surface of the cell. In some embodiments, the stimulatory agent is included in an oligomeric stimulatory reagent (e.g., streptavidin mutein oligomers conjugated to anti-CD3 Fab and anti-CD28 Fab) that can be added to the stationary phase. In some embodiments, the stimulation results in spontaneous detachment of the selected cells from the stationary phase, thus allowing collection and/or elution of the selected stimulated cells in the absence of additional processing steps to detach the cells from the stationary phase and to remove the agent used to promote detachment from the output composition of the stimulated cells. In some embodiments, the stimulation results in spontaneous detachment or release of the selected cells from the stationary phase, thus allowing collection and/or elution of the selected stimulated cells by gravity flow. In some embodiments, gravity flow relies on collecting or eluting the spontaneously detached cells from the column (e.g., stationary phase). In some embodiments, a washing step may be used, e.g., in combination with gravity flow, to elute the spontaneously detached cells from the column (e.g., stationary phase). In some embodiments, the washing step may simply involve adding cell culture medium (e.g., serum-free medium), e.g., the same medium that is present in the cell input composition prior to adding or immobilizing the cells to the stationary phase, to the column. In certain aspects, the method successfully produces an uncontaminated (e.g., free of agents used for detachment (e.g., competitors, free binders) and/or selection agents) composition of selected and stimulated cells suitable for further processing, e.g., genetic manipulation, expansion, incubation, or subsequent stimulation and/or selection rounds (e.g., polishing), within 24 hours of initiating on-column stimulation. Articles of manufacture and devices thereof are also provided.

A variety of methods are available for generating cell populations suitable for use in cell therapy (e.g., selected (enriched) and stimulated cells engineered to express a recombinant protein (e.g., a chimeric antigen receptor). However, in some aspects, these methods may require long or relatively long times to generate the cells, due at least in part to the need to perform multiple processing steps. Multiple processing steps may also result in cell stress, thus affecting the usefulness of the cells for downstream processing. Additional methods for generating cell compositions are needed.

In certain aspects, the methods provided are based on the discovery that selecting and stimulating target cells (e.g., CD3+, CD4+, or CD8+ T cells) on a stationary phase of a chromatography column promotes downregulation of the molecule (i.e., selection marker) used for cell selection, resulting in spontaneous detachment of the cells from the stationary phase. In some embodiments, the stationary phase of the chromatography column is functionalized with an agent (e.g., selection agent) capable of specifically binding to a molecule (e.g., selection marker) on the surface of the target cells. In this way, when a sample containing target cells containing a selection marker (e.g., CD3, CD4, CD8) is mixed with the stationary phase (e.g., adding the sample to the stationary phase), the target cells (e.g., CD3+, CD4+, CD8+ T cells) are indirectly immobilized on the stationary phase. In certain aspects, the target cells (e.g., T cells) are stimulated while immobilized on the stationary phase (e.g., on-column stimulation), e.g., by addition of a stimulatory agent, a stimulating reagent that includes the stimulatory agent, and/or via a stimulatory agent directly or indirectly coupled to the stationary phase. In certain embodiments, the stimulatory agent comprises an agent that activates or stimulates T cells, such as an anti-CD3/anti-CD28 antibody (e.g., Fab) agent. Thus, in some aspects, the methods and other embodiments provided are advantageous in that they simplify multiple processing steps (e.g., selection and stimulation) and/or eliminate processing steps (e.g., steps to remove selection reagents and/or selection agents used to promote detachment), allowing the simplified process to occur in the same container and/or in the same closed system, which can result in improved efficiency and sterility.

In certain aspects, the method involves the use of an oligomeric stimulatory reagent that includes a stimulatory agent capable of delivering a stimulatory signal in a target cell, such as a T cell. Exemplary oligomeric reagents include streptavidin mutein oligomers reversibly bound or conjugated to one or more antibodies or fragments thereof capable of delivering a stimulatory signal in a target cell, such as a T cell. In some embodiments, the oligomeric stimulatory reagent is a streptavidin mutein oligomer conjugated to an anti-CD3 Fab and an anti-CD28 Fab. Existing reagents that are used to stimulate T cells in vitro, for example in the absence of exogenous growth factors or in the presence of small amounts of exogenous growth factors, are known (see, for example, U.S. Pat. No. 6,352,694 (B1) and European Patent EP0700430B1). Generally, such reagents will use beads, such as magnetic beads, with a diameter of more than 1 μm, on which various binding agents, such as anti-CD3 and/or anti-CD28 antibodies, are immobilized. However, such magnetic beads may be difficult to incorporate into methods for stimulating cells, for example, under conditions required for clinical trials or therapeutic purposes, since it is necessary to ensure that these magnetic beads are substantially or completely removed before administering the engineered T cells to a subject. In some aspects, such removal, for example by exposing the cells to a magnetic field, may reduce the yield of viable cells available for cell therapy. In certain instances, such reagents, e.g., stimulating reagents containing magnetic beads, must be incubated with cells for a minimum amount of time to allow for detachment of a sufficient amount of T cells from the stimulating reagent. Furthermore, reagents such as beads are not easily compatible with column chromatography due to their physical composition.

The provided methods utilize oligomeric stimulating reagents (e.g., streptavidin mutein oligomers conjugated to anti-CD3 and anti-CD28 antibodies, e.g., Fabs) to overcome such potential limitations. For example, in some embodiments, the provided methods include adding a soluble oligomeric reagent that is not bound to a solid support (e.g., beads) to the stationary phase to initiate stimulation. In some embodiments, the provided methods can include steps to reduce or minimize the amount of residual oligomeric stimulating reagent that may be present at the end of the entire process of engineering cells for cell therapy. In some embodiments, the risk of residual reagent in output cells, e.g., engineered cells, generated or produced by the method is reduced or avoided by the use of oligomeric reagents, because the addition of a competing reagent or free binding agent can be used to dissociate (e.g., break the binding of) the oligomeric stimulating reagent from the stimulating agent in the composition containing the cells. In some embodiments, since the oligomeric stimulating reagent is soluble, it may be sufficient to reduce or remove the oligomeric stimulating reagent from the cells in the composition by one or more washing steps, without the need to add, for example, a competitive reagent or free binding agent. In some embodiments, this also means that a process that adheres to GMP standards can be more easily established than other methods, such as those that require additional measures to be taken to ensure that the final population for administration is bead-free. Thus, in some aspects, removal or separation of the oligomeric stimulating reagent from the cells, such as by adding, for example, a competitive agent or free binding agent or one or more washing steps, results in little or no cell loss compared to removal or separation of a bead-based stimulating reagent. In some aspects, the timing of reduction, removal or separation of the stimulating reagent or oligomeric stimulating reagent is not limited or is less limited than removal or separation of a bead-based stimulating reagent. Thus, in some aspects, the stimulating reagent or oligomeric stimulating reagent may be reduced, removed or separated from the cells at any time or step during the provided methods.

In certain aspects, the duration of the provided methods can be measured from the time the cells, e.g., T cells of an input cell population or input cell sample, are first contacted or exposed to stimulatory conditions (e.g., as described herein, e.g., in Sections I-C), also referred to herein as the start of incubation with a stimulator or under stimulatory conditions, e.g., exposure to a stimulatory reagent, etc. In some embodiments, the duration for the step of collecting an output population (also referred to herein as an output composition) containing stimulated target cells (e.g., CD3+, CD4+, CD8+ T cells) is measured from the start of incubation of the target cells with a stimulatory reagent (e.g., addition of or exposure to a stimulatory reagent), i.e., from the time the stimulatory reagent is added to the column. In some embodiments, the collecting step is performed by gravity flow of the cells from the column at a time point after the start of incubation, which in some examples can include one or more washes of the column to ensure recovery of cells that have spontaneously released from the column. In certain embodiments, the duration of incubation before the cells are harvested from the column is 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours, or about 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours, or less than 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours. In some embodiments, the duration of incubation before cells are eluted from the column and/or collected is 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours, or about 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours, or less than 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours or 2 hours. In some embodiments, the duration of incubation before cells are eluted from the column and/or collected is 6 hours, about 6 hours, or less than 6 hours. In some embodiments, the duration of incubation before the cells are eluted from the column and/or collected is 5 hours, about 5 hours, or less than 5 hours. In some embodiments, the duration of incubation before the cells are eluted from the column and/or collected is 4.5 hours, about 4.5 hours, or less than 4.5 hours. In some embodiments, the duration of incubation before the cells are eluted from the column and/or collected is 4 hours, about 4 hours, or less than 4 hours. In some embodiments, the duration of incubation before the cells are eluted from the column and/or collected is 3 hours, about 3 hours, or less than 3 hours. In some embodiments, the duration of incubation before the cells are eluted from the column and/or collected is 3-6 hours or about 3-6 hours. In some embodiments, the duration of incubation before the cells are eluted from the column and/or collected is 4-6 hours or about 4-6 hours. In some embodiments, the duration of incubation before cells are eluted from the column and/or collected is at or about 4-5 hours. In some embodiments, the duration of incubation with a stimulation reagent provided prior to collection from the column, such as for purposes of generating an output composition of selected and stimulated cells for use in connection with a process of genetically engineering the cells with a recombinant receptor, such as by transduction, is at or about 75%, 60%, 50%, 40%, 30%, 25%, 15% or 10% of an alternative or existing process, such as an alternative process in which selection and stimulation are performed separately and/or stimulation is not performed on the column.

It is contemplated herein that the output composition of selected and stimulated cells may be further processed. For example, the output cells may be genetically engineered to express a recombinant protein, such as a chimeric antigen receptor, and/or the output cells may be subjected to further incubation, stimulation, expansion, selection (e.g., polishing), and/or formulation. In some embodiments, the output composition of selected and stimulated cells may be further processed (e.g., manipulated, polished) to generate an output composition of engineered cells, e.g., a therapeutic cell composition useful for treating a disease in a patient.

In certain embodiments, the provided methods are performed on a sample, such as, for example, an apheresis, buffy coat, or whole blood. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is collected from a human subject. In some embodiments, the biological sample is collected from a patient suffering from a disease or condition. In some embodiments, the method is performed on a population of cells previously isolated, enriched, or selected from the sample, such as a population of CD3+ T cells. In some embodiments, the method is performed on a population of cells previously isolated, enriched, or selected from the sample, such as a population of CD4+ T cells and CD8+ T cells. In some embodiments, the sample or cells isolated from the sample may be cryopreserved.

Also provided are cells and populations prepared by the methods, e.g., pharmaceutical populations and pharmaceutical formulations, as well as kits, systems and devices for carrying out the methods. Methods of using the cells and populations prepared by the methods, e.g., therapeutic methods such as methods of adoptive cell therapy, and pharmaceutical populations for administration to a subject, are further provided.

All publications referred to in this application, including patent documents, scientific articles, and databases, are incorporated herein in their entirety for all purposes to the same extent as if each individual publication was individually incorporated herein by reference. To the extent that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

I. Methods for Selecting, Stimulating and Manipulating Cells Provided herein are methods for generating an output population (also referred to as an output composition) of cells, such as selected and stimulated T cells, such as CD3+ T cells, CD4+ T cells and CD8+ T cells, comprising steps for selecting, stimulating and collecting cells. In some embodiments, the output population of stimulated and selected cells is suitable for generating a therapeutic cell composition. In certain aspects, the method combines the selection and stimulation steps, allowing for the collection and/or elution of selected and stimulated cells that spontaneously detach from the stationary phase without the use of a competitor or free binding agent to promote detachment. Thus, the methods provided herein combine the cell selection, stimulation and collection/elution steps, and do not require separate steps to promote detachment of selected and stimulated cells from the stationary phase or purification steps to remove agents used to promote detachment (e.g., competitor and/or free binding agent). Thus, the methods reduce the number of processing steps required to generate an output composition of selected and stimulated cells suitable for downstream processing (e.g., genetic engineering, expansion, subsequent incubation, stimulation and/or selection (e.g., polishing)), thereby reducing production time, minimizing potential cell stress, and reducing the possibility of contamination. In certain embodiments, the methods generate a composition of selected and stimulated cells suitable for downstream processing within a period of time, e.g., within 24 hours. In some embodiments, the output population of selected and stimulated cells is used as an input population, or is a source for use as an input population, for a subsequent step, such as the genetic engineering described in Section IE.

In certain embodiments, the methods provided herein are used in connection with the manufacture, production or creation of cellular therapeutics. In some embodiments, the method of generating or creating an output composition, e.g., a method of generating or creating selected stimulated T cells, includes one or more steps for isolating cells from a subject and incubating the cells under stimulatory conditions. In some aspects, the output composition is used as a source of input cells for downstream processes to generate cellular therapeutics, e.g., genetically engineer cells. In some embodiments, the method includes sequential process steps in which cells, e.g., primary CD3+, CD4+, and CD8+ T cells, are isolated, e.g., selected or separated, from a biological sample in a single step, incubated under stimulatory conditions, collected or eluted, then genetically engineered to introduce a recombinant polynucleotide encoding a recombinant receptor into the cells, e.g., by transduction or transfection, and then collected, harvested, or loaded into a container, e.g., a bag or vial, as an output population of engineered cells. In some embodiments, the cells of the output population of engineered cells (e.g., a therapeutic cell composition) are reintroduced into the same subject, optionally after cryopreservation and storage of the cells. In some embodiments, the output population of engineered cells is suitable for use in therapy, e.g., autologous cell therapy.

In certain embodiments, the methods provided are used in connection with generating an output population of engineered cells expressing a recombinant receptor from an initial or input population of cells. In certain embodiments, the input population is produced, generated and/or created by providing, mixing and/or pooling the cells collected as an output composition of selected and stimulated cells by the methods provided. In some embodiments, the input population of cells contains enriched T cells, enriched CD3+ T cells, enriched CD4+ T cells and/or enriched CD8+ T cells (hereinafter also referred to as an enriched population of T cells, an enriched population of CD3+ T cells, an enriched population of CD4+ T cells and an enriched population of CD8+ T cells, respectively). In some embodiments, the input population of cells is a population of CD4+ T cells or CD8+ T cells, or is a mixed, admixed and/or pooled population of CD4+ T cells and CD8+ T cells. In some embodiments, the input population of cells is a population of CD3+ cells. In certain embodiments, the provided methods are used in conjunction with genetically engineering the selected stimulated cells, e.g., by introducing a polynucleotide encoding a recombinant protein by transduction or transfection. In certain embodiments, the methods can be used to isolate or select and stimulate cells from a biological sample, e.g., a biological sample collected, harvested, and/or obtained from a subject (e.g., whole blood, apheresis), to generate an input population of stimulated enriched T cells. In some embodiments, the provided methods can further include harvesting, collecting, and/or formulating the population of enriched T cells after manipulating, transducing, and/or culturing the cells.

In certain embodiments, the methods provided herein are practiced in connection with introducing a heterologous or recombinant polynucleotide into a cell, e.g., transducing or transfecting a cell, e.g., by a method described herein, e.g., in Sections I-E. In certain embodiments of the methods provided, the cells are incubated during or after genetic engineering of the cells for a time sufficient to allow for integration of a heterologous or recombinant polynucleotide encoding, e.g., a recombinant protein, or to allow expression of the recombinant protein. In certain embodiments, the cells are incubated for a period of time or a defined period of time, e.g., more than 18 hours or less than 4 days. In some embodiments, the manipulation step is initiated or started within a period of time from when the stimulation step is initiated or started, e.g., within 24 hours from when the cells are exposed to the stimulant. In some embodiments, the manipulation step is initiated or started within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours from when the cells are exposed to the stimulant. In some embodiments, the manipulation step is initiated or started within 2, 3, 4, 5, or 6 hours or about 2, 3, 4, 5, or 6 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 3 or 6 hours or about 3 or 6 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 4 or 6 hours or about 4 or 6 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 4 or 5 hours or about 4 or 5 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 6 hours or about 6 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 5 hours or about 5 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 4.5 hours or about 4.5 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 4 hours or about 4 hours from the time the cells are exposed to the stimuli. In some embodiments, the manipulation step is initiated or started within 3 hours or about 3 hours from the time the cells are exposed to the stimuli. In some embodiments, the incubation in the presence of the heterologous or recombinant polynucleotide, which may be contained in a virus (e.g., a viral vector), continues for a duration of 1 hour, about 1 hour, or at least 1 hour. In some embodiments, the one or more processes are carried out, at least in part, in a serum-free medium. In some embodiments, the serum-free medium is a defined or well-defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, e.g., filtered to remove inhibitors and/or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and/or attachment factors. In some embodiments, the serum-free medium comprises cytokines. In some embodiments, the serum-free medium comprises a cytokine or recombinant cytokine. In some embodiments, the serum-free medium comprises recombinant IL-2, IL-15 and/or IL-7. In some embodiments, the serum-free medium comprises glutamine. In some embodiments, the serum-free medium comprises glutamine and recombinant IL-2, IL-15 and IL-7.

In some embodiments, the methods provided are performed such that one, more, or all steps in the preparation of cells for clinical use, such as in adoptive cell therapy, are performed without exposing the cells to non-sterile conditions. In some embodiments, cell selection, stimulation, transduction, washing, and formulation are all performed within a closed, sterile system or device. In some embodiments, one or more of the steps are performed in a non-closed system or device. In some such embodiments, the cells are transferred from a closed system or device under sterile conditions to another closed system, such as by sterile transfer.

In certain embodiments, a sample and/or an isolated portion of a sample, such as a cell-containing sample in connection with one or more steps of the invention (e.g., buffy coat, enriched population of T cells), may be collected, formulated for cryoprotection, frozen (e.g., cryoprotected), and/or stored at less than 0°C, less than -20°C, or less than -70C or -80°C, or less than -70C or -80°C, before, during, or after any stage or step of the methods provided herein. In some embodiments, the cells may be stored for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or for less than 1, 2, 3, 4, 5, 6, 7, 8 weeks, or for a period of at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or for more than 8 weeks. After storage, the sample of cells or an isolated portion of the sample may be thawed and processing by the method may be resumed at the same point in the process. In certain embodiments, the cultured and/or formulated population of enriched T cells (e.g., engineered T cells) are cryoprotected and stored prior to administration to a subject, e.g., as an autologous cell therapy.

In particular embodiments, a portion of cells can be sampled or collected at any stage or step in the process, for example, cells can be collected from a population of cells (e.g., a population of T cells) while the population remains in a closed system. In certain embodiments, such cells can be analyzed for markers, traits, or characteristics, such as, but not limited to, viability, apoptosis, activation, stimulation, growth, and/or exhaustion. In some embodiments, cells are sampled or collected by an automated process (see, e.g., Section I-E-3a). In some embodiments, analysis of sampled or collected cells is automated. In certain embodiments, analysis is performed in a closed system under sterile conditions.

In some embodiments, cells or populations of cells produced and/or processed by the provided methods may be compared to cells or populations of cells processed or produced by exemplary and/or alternative processes. In certain embodiments, the alternative and/or exemplary processes may differ in one or more specific aspects, but otherwise include similar or identical attributes, aspects, steps, stages, reagents, or conditions as the aspects or aspects of the provided methods to which the exemplary or alternative processes are compared. For example, selected and stimulated cells produced by the provided methods, e.g., the output composition of selected and stimulated cells, may be compared to cells produced in a process involving separate selection and stimulation steps that require the use of a competitor or free binder to detach the selected cells from the stationary phase. In some embodiments, unless otherwise specified, the provided methods and the exemplary or alternative processes will otherwise be similar and/or identical, e.g., involving similar or identical steps of selection, enrichment, stimulation, manipulation, transfection, transduction, culture, and/or formulation. In some embodiments, unless otherwise specified, the methods and alternative processes provided select and/or enrich cells from the same or similar type of biological sample and/or process cells and/or input cells of the same cell type.

The methods provided herein reduce the time required to generate an output composition (e.g., a therapeutic cell composition) of an engineered cell. In some embodiments, the time required to generate an output composition (e.g., a therapeutic cell composition) of an engineered cell is at least 40%, 50%, 60%, 70%, 80% or 90% less than the time required by alternative processes. In some embodiments, the methods provided herein provide an output composition (e.g., a therapeutic cell composition) of an engineered cell in less than 5 days. In some embodiments, the methods provided herein provide an output cell composition (e.g., a therapeutic cell composition) of an engineered cell in at or about 4 days or at or about 96 hours. In some embodiments, the methods provided herein provide an output cell composition (e.g., a therapeutic cell composition) of an engineered cell in at or about 4 days or at or about 96 hours. In some embodiments, the methods provided herein provide an output cell composition (e.g., a therapeutic cell composition) of an engineered cell in at or about 4-5 days or at or about 96-120 hours.

A. Sample and Cell Preparation In certain embodiments, the methods provided include selecting and/or enriching cells from a biological sample. In some embodiments, the methods provided include selecting cells or populations thereof from a biological sample, e.g., a biological sample obtained or derived from a subject, such as a subject having a particular disease or condition, in need of cell therapy or to which cell therapy is administered. In some aspects, the subject is a human subject, e.g., a patient in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, processed and/or manipulated. Thus, the cells are, in some embodiments, primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject. Biological samples can be samples obtained directly from a biological source or samples that are processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample is a blood or blood-derived sample or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils or other organs, and/or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

In some instances, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some aspects, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.

In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended after washing in a wide variety of biocompatible buffers, such as, for example, Ca ⁺⁺ /Mg ⁺⁺ -free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

In some embodiments, the cell-containing sample (e.g., the apheresis product or leukapheresis product) is washed to remove one or more anticoagulants, such as heparin, that were added during apheresis or leukapheresis.

In some embodiments, a cell-containing sample (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte cell sample, an apheresis product, or a leukapheresis product) is cryopreserved and/or cryoprotected (e.g., frozen) and then thawed prior to any step of isolating, selecting, activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, formulating a population of cells, and/or administering the formulated cell population to a subject.

In some embodiments, a sample containing autologous peripheral blood mononuclear cells (PBMCs) from a subject is collected in a manner suitable to ensure adequate manufacturing quality. In one aspect, the sample containing PBMCs is derived from fractionated whole blood. In some embodiments, whole blood from a subject is fractionated by leukapheresis using centrifugal force and exploiting density differences between cell phenotypes, whereby autologous mononuclear cells (MNCs) are preferentially enriched while other cell phenotypes, such as red blood cells, are reduced in the collected cell composition. In some embodiments, autologous plasma is collected simultaneously during MNC collection, which in some aspects can allow for extended stability of the leukapheresis product. In one aspect, autologous plasma is added to the leukapheresis product to improve the buffering capacity of the leukapheresis product matrix. In some aspects, the total volume of whole blood processed to generate the leukapheresis product is at or about 2L, 4L, 6L, 8L, 10L, 12L, 14L, 16L, 18L, or 20L, or any value between any of the foregoing. In some embodiments, the volume of autologous plasma collected is at or about 10mL, 50mL, 100mL, 150mL, 200mL, 250mL, or 300mL or more, or any volume between any of the foregoing. In some embodiments, the leukapheresis product is subjected to procedures, such as washing and formulation for in-process cryopreservation, within about 48 hours of completing the leukapheresis collection. In some embodiments, the leukapheresis product is subjected to one or more washing steps, e.g., within about 2, 6, 12, 18, 24, 36, or 48 hours of completing the leukapheresis collection. In some aspects, the one or more washing steps remove anticoagulant during the leukapheresis collection, cellular waste products that may accumulate in the leukapheresis product, residual platelets, and/or cellular debris. In some embodiments, one or more buffer exchanges are performed between the one or more washing steps.

In certain embodiments, the apheresis product or leukapheresis product is cryopreserved and/or cryoprotected (e.g., frozen), as described below, and then thawed before being subjected to a cell selection or isolation step (e.g., a T cell selection or isolation step). In some embodiments, after the cryopreserved and/or cryoprotected apheresis product or leukapheresis product is subjected to a T cell selection or isolation step, no additional cryopreservation and/or cryoprotection step is performed during or between any subsequent steps, such as activating, stimulating, manipulating, transducing, transfecting, incubating, culturing, harvesting, formulating, and/or administering the formulated cell population to a subject. For example, the T cells selected from the thawed cryopreserved and/or cryoprotected apheresis product or leukapheresis product are not cryopreserved and/or cryoprotected again before being thawed for downstream processing, such as transduction.

In certain embodiments, the cryopreserved and/or cryoprotected apheresis product or leukapheresis product is banked (e.g., without cell selection prior to freezing the sample), which in some aspects can provide more flexibility for subsequent manufacturing steps. In some aspects, the cryopreserved and/or cryoprotected apheresis product or leukapheresis product is divided into multiple cryopreservation containers (such as bags) that can each be used individually or in combination in the processing of the product. In one aspect, banking the cells prior to selection can increase the cell yield of downstream processes, and banking the cells earlier can mean that they are healthier and may be easier to meet manufacturing success criteria. In another aspect, once thawed, the cryopreserved and/or cryoprotected apheresis product or leukapheresis product can be subjected to one or more different selection methods. The advantage of this approach is, among other things, to enhance the availability, efficacy and/or other aspects of the cells of the cell therapy for the treatment of a disease or condition of interest, such as in the donor of the sample and/or another recipient.

In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected and cryopreserved and/or cryoprotected at a time after the donor is diagnosed with any disease or condition, either prior to cell selection or without prior cell selection (e.g., without prior T cell selection, such as selection by chromatography). In some aspects, the time of cryopreservation is also prior to the donor receiving one or more of any initial treatment for the disease or condition, any targeted therapy or any therapy labeled for treatment for the disease or condition, or any therapy other than radiation and/or chemotherapy. In some embodiments, the sample is collected after the first recurrence of the disease after initial treatment for the disease and before the donor or subject receives a subsequent treatment for the disease. The initial treatment and/or subsequent treatment can be a therapy other than a cell therapy. In some embodiments, the collected cells may be used for cell therapy after the initial treatment and/or subsequent treatment. In one aspect, cryopreserved and/or cryoprotected samples without prior cell selection can help reduce upfront costs, such as those associated with untreated patients in randomized clinical trials who may cross over and subsequently require treatment.

In some embodiments, samples (e.g., apheresis or leukapheresis samples) are collected, cryopreserved and/or cryoprotected, either prior to cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), at a time point after second line treatment of the disease and after a second recurrence of the disease before the donor or subject undergoes a subsequent treatment for the disease. In some embodiments, patients are identified as likely to relapse after second line treatment, for example, by assessing certain risk factors. In some embodiments, the risk factors are based on disease type and/or genetic traits, such as double hit lymphoma, primary refractory cancer, or activated B cell lymphoma. In some embodiments, the risk factors are based on clinical features, such as early recurrence after first line treatment, or other poor prognostic indicators after treatment (e.g., IPI (International Prognostic Index) > 2).

In some embodiments, the sample (e.g., an apheresis or leukapheresis sample) is collected and cryopreserved and/or cryoprotected at a time before the donor or subject is diagnosed with any disease, prior to cell selection or without prior cell selection (e.g., without prior T cell selection, such as chromatographic selection). In some aspects, the donor or subject may be determined to be at risk for developing any disease. In some aspects, the donor or subject may be a healthy subject. In certain cases, the donor or subject may choose to bank or preserve the cells in case cell therapy is needed at a later stage in life, even if they are never considered to be at risk for developing any disease or being diagnosed with any disease. In some embodiments, the donor or subject may be considered at risk for developing any disease based on factors such as gene mutations, gene abnormalities, gene disruptions, family history, protein abnormalities (such as defects in protein production and/or processing), and lifestyle choices that may increase the risk of developing any disease. In some embodiments, the cells are collected as a prophylactic.

In some embodiments, cryopreserved and/or cryoprotected samples of cells (e.g., apheresis or leukapheresis samples), e.g., samples of cells that have not been subjected to prior cell selection (e.g., without prior T cell selection such as chromatographic selection), are stored or banked for a period of time greater than 12 hours, greater than 24 hours, greater than 36 hours, or greater than 48 hours, or equal to 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, samples are stored or banked for a period of time greater than 1 week, greater than 2 weeks, greater than 3 weeks, or greater than 4 weeks, or equal to 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, samples are stored or banked long term. In some aspects, the sample is greater than 1 month, greater than 2 months, greater than 3 months, greater than 4 months, greater than 5 months, greater than 6 months, greater than 7 months, greater than 8 months, greater than 9 months, greater than 10 months, greater than 11 months, greater than 1 year, greater than 2 years, greater than 3 years, greater than 4 years, greater than 5 years, greater than 6 years, greater than 7 years, greater than 8 years, greater than 9 years, greater than 10 years, greater than 11 years, greater than 12 years, greater than 13 years, greater than 14 years, greater than 15 years, greater than 16 years, greater than 17 years, greater than 18 years, greater than 19 years, greater than 20 years, greater than 25 years, greater than 30 years, greater than 35 years Stored for more than 40 years or more, or for a period equal to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years or more.

In some embodiments, an apheresis or leukapheresis sample collected from a donor is transported in a refrigerated environment to a storage or processing facility and/or cryogenically stored at a storage facility or processed at a processing facility. In some embodiments, the sample is processed prior to transport, e.g., by selecting T cells, such as CD4+ and/or CD8+ T cells. In some embodiments, such processing is performed after transport and prior to cryogenic storage of the sample. In some embodiments, processing is performed after thawing the sample following cryogenic storage.

By allowing a donor to store their cells at a stage when they, and thus their cells, have not undergone extensive treatment for a disease and/or prior to the onset or diagnosis of a disease or condition, such cells may have certain advantages for use in cell therapy compared to cells harvested after one or more treatments. For example, cells harvested before one or more treatments may be healthier, may exhibit higher levels of certain cellular activity, may grow more rapidly, and/or may be more receptive to genetic manipulation than cells that have undergone several treatments. Another example of an advantage according to the embodiments described herein may include convenience. For example, by collecting, optionally processing, and storing a donor's cells before they are needed for cell therapy, the cells are readily available when the recipient needs them later. This increases the capacity of apheresis laboratories and allows technicians more flexibility in scheduling the apheresis collection process.

Exemplary methods and systems for cryogenically storing and processing cells from a sample, such as an apheresis sample, may include those described in International Published Application No. WO2018170188. In some embodiments, the methods and systems involve collecting an apheresis before a patient requires cell therapy, then cryopreserving the apheresis sample for later use in a process of engineering cells (e.g., T cells) with a recombinant receptor (e.g., CAR). In some cases, such a process may include a process described herein. In some embodiments, an apheresis sample is collected from a subject and cryopreserved prior to subsequent T cell selection, activation, stimulation, engineering, transduction, transfection, incubation, culture, harvesting, formulation, and/or administration of a formulated cell population to the subject. In such examples, the cryopreserved apheresis sample is thawed prior to subjecting the sample to one or more selection steps, such as any described herein.

In some embodiments, a cryopreserved and/or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample), e.g., a sample of cells that has not been subjected to prior cell selection (e.g., without prior T cell selection, such as chromatographic selection), is thawed prior to use in downstream processes for producing a cell population for cell therapy, e.g., a T cell population containing CAR+ T cells. In some embodiments, such a cryopreserved and/or cryoprotected sample of cells (e.g., an apheresis or leukapheresis sample) is used in connection with the processes provided herein for engineered T cell therapy, such as CAR+ T cell therapy. In certain examples, no further step of cryopreservation is performed prior to or during the harvesting/formulation step.

B. Cell Selection by Chromatography In aspects of the methods provided herein, cells of a sample, such as T cells, are selected by chromatographic isolation, for example by column chromatography, such as affinity chromatography or gel permeation chromatography. In some embodiments, the method uses a selection agent that binds to a selection marker located on the surface of target cells, such as the cells to be isolated, selected or enriched. Such methods may also be described as (traceless) cell affinity chromatography technology (CATCH), and may include any of the methods or techniques described in PCT applications WO2013124474 and WO2015164675, which are incorporated herein by reference in their entirety.

In some embodiments, a cryopreserved and/or cryoprotected apheresis product or leukapheresis product is thawed. In some embodiments, the thawed cell composition is subjected to dilution (e.g., dilution with serum-free medium) and/or washing (washing with serum-free medium), which can remove or reduce unwanted or undesirable components in some cases. Optionally, the dilution and/or washing removes and reduces the presence of cryoprotectants, such as DMSO, contained in the thawed sample, which may otherwise adversely affect cell viability, yield, and recovery upon exposure to room temperature for extended periods of time. In some embodiments, the dilution and/or washing allows the medium of the thawed cryopreserved product to be exchanged for a serum-free medium, such as those described in Section III of this specification or PCT/US2018/064627, which is incorporated herein by reference.

In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T-Cell Expansion Basal Medium (ThermoFisher). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion and/or activation of cells (e.g., T cells), such as those provided by additional supplements (e.g., OpTmizer™ T-cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium comprises a serum replacement supplement, such as an immune cell serum replacement, e.g., ThermoFisher, #A2596101, CTS™ Immune Cell Serum Replacement, or Smith et al. Clin Transl Immunology. 2015 Jan;4(1):e31. In some embodiments, the serum-free medium further comprises a free amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15.

In some embodiments, cells, e.g., target cells, have or express a selection marker, as described herein, on the cell surface such that the cells to be isolated, selected or enriched are defined by the presence of at least one common specific receptor molecule. In some embodiments, a sample containing target cells may also contain excess cells that lack the selection marker. For example, in some embodiments, T cells are selected, isolated or enriched from a sample containing multiple cell types, e.g., red blood cells or B cells.

In some embodiments, the selection agent is included in the chromatography column, e.g., directly or indirectly bound to a chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent is present on the chromatography matrix (e.g., stationary phase) at the time the sample is added to the column. In some embodiments, the selection agent has the ability to be indirectly bound to the chromatography matrix (e.g., stationary phase) via a reagent, e.g., a selection reagent described herein, e.g., in Section II-A. In some embodiments, the selection agent is covalently or non-covalently bound to the column stationary phase. In some embodiments, the selection agent is reversibly immobilized on the chromatography matrix (e.g., stationary phase). In some cases, the selection agent is immobilized on the chromatography matrix (e.g., stationary phase) by a covalent bond. In some aspects, the selection agent is reversibly immobilized on the chromatography matrix (e.g., stationary phase) by a non-covalent bond.

In some embodiments, the selection agent may be present at the time the sample is added to the chromatography column (e.g., stationary phase), either directly (e.g., covalently or non-covalently) or indirectly bound via a selection reagent, for example, to the chromatography matrix (e.g., stationary phase). Thus, upon addition of the sample, the target cells may be bound by the selection agent and immobilized on the chromatography matrix (e.g., stationary phase) of the column. Alternatively, in some embodiments, the selection agent may be added to the sample. In this way, the selection agent binds to the target cells (e.g., T cells) in the sample, and the sample may then be added to the chromatography matrix (e.g., stationary phase) containing the selection agent. In this case, the selection agent already bound to the target cells binds to the selection agent, thereby immobilizing the target cells on the chromatography matrix (e.g., stationary phase). In some embodiments, the selection agent binds to a selection agent, e.g., as described in Sections II-A and II-B herein, via a binding partner C, as described herein, contained in the selection agent.

In some aspects, a selection agent is added to the sample. In certain embodiments, the selection agent has a binding site B that specifically binds to a receptor molecule (e.g., a selection marker) on the surface of a cell, e.g., a target cell. See, e.g., Section II-B et seq. In some aspects, the selection agent also includes a binding partner C that can specifically and reversibly bind to binding site Z of the selection reagent.

In certain aspects, the selection reagent may also contain two or more binding sites Z that can be bound by a binding partner C, thereby resulting in multimerization of the receptor-binding reagent. Thus, the selection reagent as used herein may also be a multimerization reagent. The selection reagent may be, for example, streptavidin, a streptavidin mutein, avidin, an avidin mutein, or mixtures thereof. In some aspects, different chromatographic matrices may be coupled to different selection reagents and layered into columns to form a multicomponent system for separation.

In some embodiments, two or more selection agents are associated, e.g., reversibly or irreversibly bound, to the selection agent, e.g., via one or more binding sites Z present on the selection agent. In some cases, this results in the selection agents being closely spaced relative to one another such that an avidity effect can occur when a target cell bearing (at least two copies of) a cell surface molecule (e.g., a selection marker) is contacted with a selection agent capable of binding to the molecule (e.g., a selection marker).

In some embodiments, two or more different selection substances, which are the same, i.e., have the same selection marker binding specificity, can be reversibly bound to the selection reagent. In some embodiments, it is possible to use at least two different selection substances, and possibly three or four different selection substances, that bind different selection markers. In some aspects, each of the at least two selection substances can bind to a different molecule (e.g., selection marker), e.g., a first molecule, a second molecule, etc. In some cases, the different molecules (e.g., selection markers), e.g., different cell surface molecules, can be present on the same target cell. In another example, the different molecules (e.g., selection markers), e.g., different cell surface molecules, can be present on different target cells present in the same cell population. In some cases, a third, fourth, etc. selection substance can be associated with the same reagent, each of which further contains a different binding site.

In some embodiments, the two or more different selection substances contain the same binding partner C. In some embodiments, the two or more different selection substances contain different binding partners. In some aspects, the first selection substance can have a binding partner C1 capable of specifically binding to a binding site Z1 present on the selection reagent, and the second selection substance can have a binding partner C2 capable of specifically binding to a binding site Z1 or a binding site Z2 present on the selection reagent. Thus, in some examples, the multiple binding sites Z contained in the selection reagent include binding sites Z1 and Z2, which are capable of reversibly binding to the binding partners C1 and C2 contained in the selection substance, respectively. In some embodiments, C1 and C2 are the same, and/or Z1 and Z2 are the same. In another aspect, one or more of the multiple binding sites Z can be different. In another example, one or more of the multiple binding partners C can be different. It is within the level of one of ordinary skill in the art to choose any combination of different binding partners C that is compatible with the selection reagent containing the binding site Z, as long as each of the binding partners C can interact, e.g., specifically bind, to one of the binding sites Z.

In certain embodiments, a sample, e.g., a sample containing cells and a selection agent, is added to or contacted with a chromatography matrix containing an attached or immobilized selection reagent. In certain aspects, the selection reagent has multiple binding sites Z that specifically bind to the binding partner C of the selection agent. In certain aspects, the selection agent binds to the selection reagent through an interaction between the binding partner C and the binding site Z. Thus, in some embodiments, cells, e.g., target cells, are immobilized on the chromatography matrix via a complex formed by one or more binding sites Z of the selection reagent and the binding site Z of the selection agent. In further aspects, cells, e.g., target cells, can be depleted from the sample, e.g., by rinsing, releasing, or washing away the remaining sample from the chromatography matrix. In certain aspects, the selection agent can be included in the sample containing the cells, or can be applied to or contacted with the chromatography matrix, e.g., before adding the sample to the chromatography matrix, to bind to the attached selection reagent or multimerization reagent.

In some embodiments, the reversible bond formed between binding partner C and binding site Z can be disrupted by a competitor and/or free binder. In some embodiments, the competitor and/or free binder can be biotin, a biotin derivative, a biotin analog, or a streptavidin-binding peptide capable of competing for binding with binding partner C for one or more binding sites Z. In some embodiments, unlike binding partner C and the competitor and/or free binder, the competitor and/or free binder exhibits a higher binding affinity for one or more binding sites Z than the affinity of the binding partner. In certain aspects of any of the methods provided herein, the addition of a competitor and/or free binder to the stationary phase of a chromatography column to disrupt binding of the selection agent to the selection reagent is not necessary to detach target cells (T cells) from the chromatography matrix (e.g., stationary phase).

In some embodiments, the sample may be depleted of cells, e.g., target cells of the sample, e.g., by rinsing, releasing, or washing away remaining sample from the chromatography matrix (e.g., stationary phase). In some embodiments, one or more (e.g., 2, 3, 4, 5, 6) washing steps are used to remove unbound cells and debris from the chromatography matrix (e.g., stationary phase). In some embodiments, at least two washing steps are performed. In some embodiments, the sample is allowed to permeate the matrix for at least or about 5, 10, 15, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes before performing one or more washing steps. In some embodiments, a wash step is performed at, or about, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 or 120 minutes after application of the sample to the chromatographic column (e.g., stationary phase). In some embodiments, a wash step is performed at, or about, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes after application of the sample to the chromatographic column (e.g., stationary phase). In some embodiments, one or more wash steps are performed within or about 120, 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 minutes following addition of the sample to the chromatographic column (e.g., stationary phase). In some embodiments, one or more wash steps are performed within or about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 minutes following addition of the sample to the chromatographic column (e.g., stationary phase). In some embodiments, the addition of the sample to the chromatographic column (e.g., stationary phase) is followed by one or more wash steps within 5-60 minutes or within about 5-60 minutes. In some embodiments, the addition of the sample to the chromatographic column (e.g., stationary phase) is followed by one or more wash steps within 5-50 minutes or within about 5-50 minutes. In some embodiments, the addition of the sample to the chromatographic column (e.g., stationary phase) is followed by one or more wash steps within 5-40 minutes or within about 5-40 minutes. In some embodiments, the addition of the sample to the chromatographic column (e.g., stationary phase) is followed by one or more wash steps within 5-30 minutes or within about 5-30 minutes. In some embodiments, the addition of the sample to the chromatographic column (e.g., stationary phase) is followed by one or more wash steps within 5-20 minutes or within about 5-20 minutes. In some embodiments, the addition of the sample to the chromatographic column (e.g., stationary phase) is followed by one or more wash steps within 5-10 minutes or within about 5-10 minutes. In some embodiments, the addition of the sample to the chromatography column (e.g., stationary phase) is followed by one or more wash steps within or about 10-60 minutes. In some embodiments, the addition of the sample to the chromatography column (e.g., stationary phase) is followed by one or more wash steps within or about 20-60 minutes. In some embodiments, the addition of the sample to the chromatography column (e.g., stationary phase) is followed by one or more wash steps within or about 30-60 minutes. In some embodiments, the addition of the sample to the chromatography column (e.g., stationary phase) is followed by one or more wash steps within or about 40-60 minutes. In some embodiments, the addition of the sample to the chromatography column (e.g., stationary phase) is followed by one or more wash steps within or about 50-60 minutes.

In some embodiments, multiple rounds of cell selection steps are performed, where a fraction positively or negatively selected in one step is subjected to another selection step, e.g., a subsequent positive or negative selection. In certain embodiments, methods, techniques and reagents for selection, isolation and enrichment are described, e.g., in PCT application WO2015164675, which is incorporated herein by reference in its entirety.

In some embodiments, a single selection step can be used to isolate target cells (e.g., CD3+ T cells) from a sample. In some embodiments, the single selection step can be performed on a single chromatography column. In some examples, a single selection step can simultaneously deplete cells expressing multiple markers. Similarly, multiple cell types can be simultaneously positively selected. In certain embodiments, a selection step is repeated and/or performed more than once, where a fraction positively or negatively selected in a step is subjected to the same selection step, e.g., repeated positive or negative selection. In some examples, a single selection step is repeated and/or performed more than once, e.g., to increase the purity of selected cells and/or to further remove and/or deplete negatively selected cells from the negatively selected fraction. In certain embodiments, one or more selection steps are performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In certain embodiments, the one or more selection steps are performed and/or repeated 1-10 times, 1-5 times, or 3-5 times. In some embodiments, two selection steps are performed.

Cell selection can be performed using one or more chromatography columns. In some embodiments, one or more chromatography columns are included in a closed system. In some embodiments, the closed system is an automated closed system, e.g., requiring little or no user (e.g., human) input. In some embodiments, cell selection is performed sequentially (e.g., sequential selection techniques). In some embodiments, one or more chromatography columns are arranged sequentially. For example, a first column can be oriented such that the output (e.g., eluate) of the column is fed to a second chromatography column, e.g., through a connected tubing system. In some embodiments, multiple chromatography columns can be arranged sequentially. In some embodiments, cell selection is achieved by performing sequential positive and negative selection steps, subsequent steps subjecting the positive and/or negative fractions from the previous step to further selection, and the entire process can be performed in the same set of tubes or tubing. In some embodiments, a first selection is performed to enrich for one of the CD4+ or CD8+ populations, and a sample containing target cells is subjected to a sequential selection in which the cells not selected in the first selection are used as a cell source for the second selection to enrich for the other of the CD4+ or CD8+ populations. In some embodiments, one or more further selections can be performed to enrich for subpopulations of one or both of the CD4+ or CD8+ populations, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+. In some embodiments, a first selection is performed to enrich for the CD3+ population, and a sample containing target cells is subjected to a sequential selection in which the selected cells are used as a cell source for the second selection to enrich for the CD3+ population. In some embodiments, a first selection is performed to enrich the CD3+ population onto a first stationary phase (e.g., in a first chromatographic column), and a second selection is performed to enrich the CD3+ population onto a second stationary phase (e.g., in a second chromatographic column), in which the sample containing the target cells is subjected to a sequential selection in which the first and second stationary phases are sequentially disposed, in which the flow-through fraction containing unbound cells is used as the cell source. In some embodiments, one or more further selections can be performed to enrich for subpopulations of the CD3+ population, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+. In some embodiments, the first selection is performed to enrich for the CD3+ population, and the second selection is performed to enrich for the CD4+ population by subjecting the sample containing the target cells to a sequential selection in which the selected cells are used as a cell source. In some embodiments, one or more further selections can be performed to enrich for subpopulations of the CD3+CD4+ population, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+. In some embodiments, the first selection is performed to enrich for the CD3+ population, and the second selection is performed to enrich for the CD8+ population by subjecting the sample containing the target cells to a sequential selection in which the selected cells are used as a cell source. In some embodiments, one or more further selections can be performed to enrich for subpopulations of the CD3+CD8+ population, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+. In some aspects, it is contemplated that specific subpopulations of T cells (e.g., CD3+ cells), such as cells that are positive for or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are selected by positive or negative sequential selection techniques.

In some embodiments, cell selection is performed in parallel (e.g., parallel selection techniques). In some embodiments, one or more chromatography columns are arranged in parallel. For example, two or more columns can be arranged such that a sample is loaded onto two or more columns simultaneously via a tube that allows the sample to be added to each column without the need for the sample to pass through the first column. For example, using parallel selection techniques, cell selection can be accomplished by performing positive and/or negative selection steps simultaneously, e.g., in a closed system where the entire process is performed within the same tube or set of tubes. In some embodiments, a sample containing target cells is subjected to parallel selection, where the sample is loaded onto two or more chromatography columns, each column achieving selection of a cell population. In some embodiments, two or more chromatography columns achieve selection of CD3+, CD4+, or CD8+ populations individually. In some embodiments, two or more chromatography columns, including affinity chromatography or gel permeation chromatography, independently achieve selection of the same cell population. For example, two or more chromatography columns may achieve selection of CD3+ cells. In some embodiments, two or more chromatographic columns, including affinity chromatography or gel permeation chromatography, independently achieve the selection of different cell populations. For example, two or more chromatographic columns may independently achieve the selection of CD3+ cells, CD4+ cells and CD8+ cells. In some embodiments, one or more additional rounds of selection, for example using sequential selection techniques, can be achieved to enrich subpopulations of one or all of the cell populations selected through parallel selection. For example, the selected cells can be further selected for central memory T (T _CM ) cells, naive T cells and/or cells that are positive for or express high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and/or CD45RO+). In some embodiments, a sample containing target cells is subjected to parallel selection, which is achieved on two or more columns to enrich for CD3+ populations. In some embodiments, one or more additional rounds of selection can be accomplished to enrich for subpopulations of the CD3+ population, such as central memory T (T _CM ) cells, naive T cells, and/or cells positive for or expressing high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+). In some embodiments, a sample containing target cells is subjected to parallel selection, where selection is accomplished independently on two or more columns to enrich for CD3+ and CD4+ populations. In some embodiments, one or more additional rounds of selection can be performed to enrich for subpopulations of the CD3+ and CD4+ populations, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive for or express high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+). In some embodiments, the sample containing the target cells is subjected to parallel selections performed to enrich for the CD3+ and CD8+ populations. In some embodiments, one or more additional rounds of selection can be performed to enrich for subpopulations of the CD3+ and CD8+ populations, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive for or express high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+). In some embodiments, the sample containing the target cells is subjected to parallel selections that are performed to enrich for the CD4+ and CD8+ populations. In some embodiments, one or more additional rounds of selection can be performed to enrich for subpopulations of the CD4+ and CD8+ populations, such as central memory T (T _CM ) cells, naive T cells, and/or cells that are positive or express high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+). In some aspects, it is envisioned that specific subpopulations of T cells (e.g., CD3+, CD4+, CD8+ T cells), such as cells that are positive or express high levels of one or more surface markers (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells), are selected by positive or negative parallel selection techniques. In some embodiments, serial and parallel selection techniques can be used in combination.

In some embodiments, two columns are used for parallel selection. In some embodiments, the two columns select for the same cell type (e.g., the same selection marker). In some embodiments, the two columns each select for CD3+ T cells.

In general, the binding capacity of the stationary phase (e.g., selection resin) affects the amount of stationary phase required to select a certain number of target moieties, e.g., target cells, such as T cells. The binding capacity, e.g., the number of target cells that can be immobilized per mL of stationary phase (e.g., selection resin), can be used to determine or control the number of target cells captured in one or more columns. One or more chromatography columns can be used for on-column cell selection and cell stimulation disclosed herein. When multiple columns are used, they can be arranged sequentially, in parallel, or in any suitable combination thereof. Thus, the binding capacity of the stationary phase (e.g., selection resin) can be used to standardize the amount of reagents in a single column approach or for each column in a multiple column approach.

In some embodiments, the binding capacity of a stationary phase as used herein is the maximum number of target cells that will be bound to the stationary phase under given solvent and cell concentration conditions when an excess of target cells is loaded onto the stationary phase. In some embodiments, the binding capacity is 100 million ± 25 million target cells (e.g., T cells) or about 100 million ± 25 million target cells per mL of stationary phase. In some embodiments, the static binding capacity of a stationary phase (e.g., selection resin) disclosed herein is in the range of about 75 million to about 125 million target cells per mL of stationary phase. In one aspect, the binding capacity of a stationary phase used herein for on-column cell selection and cell stimulation is the static binding capacity. In some embodiments, the static binding capacity is the maximum amount of cells that can be immobilized on the stationary phase under, for example, constant solvent and cell concentration conditions. In some embodiments, the static binding capacity of a stationary phase (e.g., selection resin) disclosed herein is in the range of about 50 million to about 100 million target cells per mL of stationary phase. In some embodiments, the static binding capacity is at or about 100 million ± 25 million target cells (e.g., T cells) per mL of stationary phase. In some embodiments, the static binding capacity of the stationary phases (e.g., selection resins) disclosed herein ranges from about 75 million to about 125 million target cells per mL of stationary phase. In some embodiments, the static binding capacity of the stationary phase (e.g., a selection resin) is from about 10 million to about 20 million target cells per mL of stationary phase, from about 20 million to about 30 million, from about 30 million to about 40 million, from about 40 million to about 50 million, from about 50 million to about 60 million, from about 60 million to about 70 million, from about 70 million to about 80 million, from about 80 million to about 90 million, from about 90 million to about 100 million target cells per mL of stationary phase. billion, about 110 million to about 120 million, about 120 million to about 130 million, about 130 million to about 140 million, about 140 million to about 150 million, about 150 million to about 160 million, about 160 million to about 170 million, about 170 million to about 180 million, about 180 million to about 190 million, or about 190 million to about 200 million.

In some embodiments, the binding capacity of a stationary phase as used herein is the number of target cells that will bind to the stationary phase under given flow conditions before significant breakthrough of unbound target cells occurs. In one aspect, the binding capacity of a stationary phase as used herein for on-column cell selection and cell stimulation is the dynamic binding capacity, i.e., the binding capacity under operating conditions in a packed chromatography column during sample application. In some embodiments, the dynamic binding capacity is determined by loading a sample containing a known concentration of target cells and monitoring the flow-through fraction. Target cells will bind to the stationary phase up to a certain breakthrough point, after which unbound target cells will flow through the column. In some embodiments, the dynamic binding capacity is 100 million ± 25 million or about 100 million ± 25 million target cells (e.g., T cells) per mL of stationary phase. In some embodiments, the dynamic binding capacity of a stationary phase (e.g., selection resin) disclosed herein is 75 million to about 125 million or about 75 million to about 125 million target cells per mL of stationary phase. In some embodiments, the dynamic binding capacity of the stationary phase (e.g., selection resin) disclosed herein is in the range of about 50 million to about 100 million target cells per mL of stationary phase. In some embodiments, the dynamic binding capacity of the stationary phase (e.g., selection resin) is in the range of about 10 million to about 20 million target cells per mL of stationary phase, about 20 million to about 30 million target cells per mL of stationary phase, about 30 million to about 40 million target cells per mL of stationary phase, about 40 million to about 50 million target cells per mL of stationary phase, about 50 million to about 60 million target cells per mL of stationary phase, about 60 million to about 70 million target cells per mL of stationary phase, about 70 million to about 80 million target cells per mL of stationary phase, about 80 million to about 90 million target cells per mL of stationary phase, about 90 million to about 100 million target cells per mL of stationary phase, about 10 million to about 20 million target cells per mL of stationary phase, about 20 million to about 30 million target cells per mL of stationary phase, about 30 million to about 40 million target cells per mL of stationary phase, about 40 million to about 50 million target cells per mL of stationary phase, about 50 million to about 60 million target cells per mL of stationary phase, about 60 million to about 70 million target cells per mL of stationary phase, about 70 million to about 80 million target cells per mL of stationary phase, about 80 million to about 90 million target cells per mL of stationary phase, about 10 million to about 20 million target cells per mL of stationary phase, about 10 million to about 20 million target cells per mL of stationary phase, about 10 million to about 30 million target cells per mL of stationary phase, about 10 million to about 30 million target cells per mL of stationary phase, about 10 million to about 30 million target cells billion, about 110 million to about 120 million, about 120 million to about 130 million, about 130 million to about 140 million, about 140 million to about 150 million, about 150 million to about 160 million, about 160 million to about 170 million, about 170 million to about 180 million, about 180 million to about 190 million, or about 190 million to about 200 million.

In some embodiments, the stationary phase is 20 mL. In some embodiments, the stationary phase has a binding capacity of 2 billion ± 500 million cells.

Any material may be used as a chromatography matrix (e.g., stationary phase). In general, suitable chromatographic materials are essentially non-toxic, i.e., not detrimental to cell viability, when used under desired conditions, e.g., as a packed chromatographic column. In some embodiments, the stationary phase remains in a predetermined location, e.g., at a predetermined position, even as the position of the sample changes. Thus, in some embodiments, the stationary phase is the portion of the chromatographic system through which the mobile phase flows (either by flow-through or batch mode) and partitioning of components contained in the liquid phase (dissolved or dispersed) between the phases occurs.

In some embodiments, the chromatography matrix has the form of a solid or semi-solid phase, while the sample containing the target cells to be isolated/separated is in a fluid phase. The chromatography matrix can be a particulate material (of any suitable size and geometry) or a monolithic chromatography material, including a paper substrate or a paper membrane. Thus, in some aspects, the chromatography can be column chromatography or planar chromatography. In some embodiments, not only standard chromatography columns, but also columns capable of bidirectional flow, such as PhyTip® columns available from PhyNexus, Inc. (San Jose, Calif., USA), can be used for column-based/flow-through mode-based methods. Thus, in some cases, pipette tips or columns capable of bidirectional flow are also included among the chromatography columns useful in the present methods. In some cases, for example, when certain matrix materials are used, the particulate matrix can have an average particle size of, for example, about 5 μm to about 200 μm, or about 5 μm to about 400 μm, or about 5 μm to about 600 μm. In some aspects, the chromatography matrix can be or include, for example, a polymeric resin or a metal oxide or semi-metal oxide. In some aspects, for example when planar chromatography is used, the matrix material can be any material suitable for planar chromatography, such as a conventional cellulose-based or organic polymer-based membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or a silica-coated glass plate. In one embodiment, the chromatography matrix/stationary phase is a non-magnetic or non-magnetizable material.

In some embodiments, non-magnetic or non-magnetizable chromatographic stationary phases suitable in the present methods include derivatized silica or cross-linked gels. In some aspects, the cross-linked gels can be based on natural polymers, e.g., classes of polymers that exist in nature. For example, natural polymers that can be the basis of the chromatographic stationary phase are polysaccharides. In some cases, each polysaccharide is typically cross-linked. Examples of polysaccharide matrices include, but are not limited to, agarose gels (e.g., Superflow™ agarose or Sepharose™ materials, e.g., Superflow™ Sepharose™, which are commercially available in a variety of bead sizes and pore sizes) or gels of cross-linked dextran. A further example is a particulate cross-linked agarose matrix to which dextran is covalently attached, which is commercially available as Sephadex™ or Superdex™, both from GE Healthcare (in a variety of bead sizes and in a variety of pore sizes). Another example of such a chromatographic material is Sephacryl™, which is also commercially available from GE Healthcare in a variety of bead sizes and pore sizes.

In some embodiments, the crosslinked gel may be based on synthetic polymers, such as classes of polymers that do not exist in nature. In some aspects, such synthetic polymers that are the basis of the chromatographic stationary phase are polymers that have polar monomeric units and therefore are themselves polar. In some cases, such polar polymers are therefore hydrophilic. Hydrophilic molecules are also called oleophobic, and in some aspects contain moieties that can form dipole-dipole interactions with water molecules. In general, hydrophobic molecules, also called oleophilic, have a tendency to move away from water.

In general, the chromatographic method is fluid chromatography, typically liquid chromatography. In some aspects, chromatography can be performed in a flow-through mode, where a fluid sample containing cells, e.g., target cells, is applied, e.g., by gravity flow or by a pump, to one end of a column containing a chromatography matrix, and the fluid sample exits the column at the other end of the column. In addition, chromatography can be performed in an "up and down" mode, where a fluid sample containing cells to be isolated is applied, e.g., by a pipette to one end of a column containing a chromatography matrix packed in a pipette tip, and the fluid sample enters the chromatography matrix/pipette tip and exits the other end of the column. Alternatively, chromatography can also be performed in a batch mode, where a chromatographic material (stationary phase) is incubated, e.g., under shaking, rotation or repeated contact, with a sample containing cells, and the fluid sample is removed, e.g., with a pipette.

In some aspects, any material may be utilized as a chromatography matrix in the context of the present invention, so long as it is suitable for chromatographic isolation, e.g., cell selection. In certain aspects, suitable chromatography materials are at least non-toxic or essentially non-toxic, e.g., not detrimental to cell viability, when used in a packed chromatography column under the desired conditions for cell isolation and/or cell separation. In some aspects, the chromatography matrix remains in a predetermined location, typically in a predetermined position, while the location of the sample to be separated and the components contained therein changes. Thus, in some aspects, the chromatography matrix is a "stationary phase."

Typically, the respective chromatographic matrix has a solid or semi-solid phase form, while the sample containing the target cells to be isolated/separated is in a fluid phase. The mobile phase used to accomplish the chromatographic separation is likewise in a fluid phase. The chromatographic matrix can be a particulate material (of any suitable size and shape), or a monolithic chromatographic material, including a paper substrate or a membrane. Thus, the chromatography can be both column chromatography and planar chromatography. In addition to standard chromatographic columns, columns or pipette tips that allow bidirectional flow can be used for column-based/flow-through mode-based chromatographic separation of cells as described herein. In some aspects, a particulate matrix material is used, which can have an average particle size of, for example, about 5 μm to about 200 μm, or about 5 μm to about 400 μm, or about 5 μm to about 600 μm. In some aspects, planar chromatography is used, and the matrix material can be any material suitable for planar chromatography, such as a conventional cellulosic or organic polymeric membrane (e.g., a paper membrane, a nitrocellulose membrane, or a polyvinylidene difluoride (PVDF) membrane) or a silica-coated glass plate.

In some aspects, the chromatographic matrix/stationary phase is a non-magnetic or non-magnetizable material. Such materials may include derivatized silica or cross-linked gels. Cross-linked gels (typically manufactured in bead form) may be based on natural polymers such as cross-linked polysaccharides. Suitable examples include, but are not limited to, agarose gels or cross-linked dextran gels. Cross-linked gels may also be based on synthetic polymers, i.e., polymer classes that do not occur in nature. Typically, such synthetic polymers that are the basis of chromatographic stationary phases for cell separations are polymers that have polar monomeric units and are therefore themselves polar.

Examples of suitable synthetic polymers are polyacrylamides, styrene-divinylbenzene gels, and copolymers of acrylates and diols or acrylamides and diols. An example is polymethacrylate gel commercially available as Fractogel®. A further example is copolymers of ethylene glycol and methacrylate commercially available as Toyopearl®. In some embodiments, the chromatographic stationary phase may also include natural and synthetic polymeric components, such as composite matrices or composites or copolymers of polysaccharides and agarose or polysaccharides and N,N'-methylenebisacrylamide, such as polyacrylamide/agarose composites. An example of a copolymer of dextran and N,N'-methylenebisacrylamide is the Sephacryl® series of materials mentioned above. Derivatized silica may include silica particles coupled to synthetic or natural polymers. Examples of such embodiments include, but are not limited to, polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, and poly(N-isopropylacrylamide)-grafted silica.

The chromatography matrices utilized in the present invention are, in some embodiments, gel filtration (also known as size exclusion) matrices. Gel filtration can be characterized by the property that they are designed to be at least essentially free from interactions with the cells to be separated. Thus, the gel filtration matrix allows for separation of cells or other biological entities as defined herein primarily based on their size. Each chromatography matrix is typically a particulate porous material as described above. The chromatography matrix can have a certain exclusion limit, typically defined in terms of molecular weight, above which molecules are completely blocked from entering the pores. The respective molecular weights defining the size exclusion limit can be selected to be below a weight corresponding to the weight of the target cells (or biological entities) to be isolated. In such embodiments, the target cells are prevented from entering the pores of the size exclusion chromatography matrix. Similarly, the stationary phase, which is an affinity chromatography matrix, can have pores that are smaller in size than the size of the selected target cells. In an illustrative embodiment, the affinity chromatography matrix and/or the gel filtration matrix has an average pore size of 0 to about 500 nm.

Other components present in the sample, such as the stimulating substance and/or the stimulating reagent (e.g., oligomeric stimulating reagent), may have a size below the exclusion limit of the pores, which can then enter the pores of the size-exclusion chromatography matrix. Of those components that can enter the pore volume partially or completely, the larger molecules with less access to the pore volume will usually elute first, while the smallest molecules will elute last. In some embodiments, the exclusion limit of the size-exclusion chromatography matrix is selected to be below the maximum width of the target cells. Thus, the components that can access the pore volume will usually remain in/on the size-exclusion chromatography matrix longer than the target cells. The target cells can then be collected in the eluate of the chromatography column separately from other objects/components of the sample. Thus, components such as the stimulating reagent elute from the gel filtration matrix at a later time than the target cells. This separation effect will be further increased if the gel permeation matrix contains (usually covalently bound thereto) a selection reagent that contains a binding site, such as binding site Z, that can bind to a reagent, such as the selection reagent and/or the competitive reagent, present in the sample. The selection agent and/or the competing reagent will be bound by the binding site Z of the affinity reagent and thereby immobilized on the gel permeation matrix. The method is typically carried out in a removal cartridge, and in some embodiments, the methods, combinations and kits of the invention include and/or use such a gel filtration matrix. Thus, in each method, cells are separated on the basis of size.

The chromatography matrix used in the present invention may also include a magnetically attractable object, such as one or more magnetically attractable particles or a magnetic fluid. Each magnetically attractable particle may include a selection reagent having a binding site (e.g., a selection substance) capable of binding to and immobilizing a target cell on the chromatography matrix. The magnetically attractable particles may contain diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic materials. Superparamagnetic materials produce an induced magnetic field in response to a magnetic field, but do not become permanently magnetized. Magnetic particles based on iron oxide are commercially available, for example, from Dynal Biotech as Dynabeads®, from Miltenyi Biotec as magnetic MicroBeads, from CPG Inc. as magnetic porous glass beads, and from a variety of other sources, such as Roche Applied Science, BIOCLON, BioSource International Inc., micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc., to name just a few. Magnetic nanoparticles based on superparamagnetic Co and FeCo and ferromagnetic Co nanocrystals are described, for example, in Huetten, A. et al. (J. Biotech. (2004), 112, 47-63). However, in some embodiments, the chromatography matrix used in the present invention does not contain any magnetically attractable objects.

Selection Agent As mentioned above, in certain aspects, the methods provided herein use a selection agent. In some embodiments, the agent described in section II-B is a selection agent. In some embodiments, the selection agent binds to a molecule on the cell surface, e.g., a cell surface molecule. In some examples, the cell surface molecule is a selection marker. In some embodiments, the selection agent has the ability to specifically bind to a selection marker expressed by one or more of the cells in the sample. In some embodiments, specific binding to a molecule, such as a cell surface molecule or cell surface receptor, throughout this disclosure does not necessarily mean that the agent only binds to such a molecule. For example, an agent that specifically binds to a molecule may also bind to other molecules, generally with much lower affinity, as determined, for example, by immunoassay, BIAcore®, KinExA3000 instrument (Sapidyne Instruments, Boise, Idaho) or other assay. In some cases, the ability of an agent to bind to a target molecule under specific binding conditions is such that its affinity or avidity is at least 5 times, e.g., at least 10, 20, 30, 40, 50, 100, 250 or 500 times, or even at least 1000 times, the average affinity or avidity of the agent for a random collection of peptides or polypeptides of sufficient statistical size.

In some embodiments, cells, e.g., target cells (e.g., T cells), have or express a molecule, e.g., a selection marker, on the cell surface such that the cells to be selected are defined by the presence of at least one common specific molecule (e.g., selection marker). In some embodiments, a sample containing target cells may also contain extra cells that lack the molecule (e.g., selection marker). For example, in some embodiments, T cells may be selected from a sample containing multiple cell types, e.g., red blood cells or B cells. Selection marker and receptor molecule may be used interchangeably herein as terms referring to cell surface molecules.

In some embodiments, the selection marker (e.g., receptor molecule) located on the cell surface, e.g., on the target cell surface, can be any molecule, so long as it remains covalently or non-covalently bound to the cell surface during the chromatographic separation process in the methods of the invention. The selection marker (e.g., receptor molecule) is a molecule that can be targeted by a selection agent. In some embodiments, the selection marker is a peptide or protein, e.g., a membrane receptor protein. In some embodiments, the selection marker is a lipid, polysaccharide, or nucleic acid. A selection marker (e.g., receptor molecule) that is a protein can be a peripheral membrane protein or an integral membrane protein, which in some embodiments can have one or more domains that span the membrane. In certain embodiments, the selection marker is a surface protein of an immune cell, e.g., CD3, CD4, or CD8. In some embodiments, the selection marker can be an antigen that defines a desired cell population or cell subpopulation, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, CD4+ T cells, or CD8+ T cells).

In some aspects, the cell surface molecule, e.g., a selection marker, can be an antigen that defines a desired cell population or cell subpopulation, e.g., a population or subpopulation of blood cells, e.g., lymphocytes (e.g., T cells, T helper cells, e.g., CD3+ T cells, CD8 T cells, CD4+ T helper cells, B cells, or natural killer cells), monocytes, or stem cells, e.g., CD34-positive peripheral stem cells or Nanog- or Oct-4-expressing stem cells. In some embodiments, the selection marker can be a marker expressed on the surface of a T cell or T cell subset, e.g., CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and/or CD45RO. Examples of T cells include cells such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). A specific example of a Treg is a CD4 CD25 CD45RA Treg cell, and a specific example of a memory T cell is a CD62L CD8+ specific central memory T cell.

As noted above, in some embodiments, the selection agent has or contains a binding site B. In certain embodiments, binding site B is monovalent. In some aspects, the monovalent binding site B is or contains a monovalent antibody fragment or a proteinaceous binding molecule with immunoglobulin-like function, an aptamer, or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single chain Fv fragments (scFv), including bivalent single chain Fv fragments. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.J., et al., Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, one or more binding sites of the selection agent may be bivalent proteinaceous artificial binding molecules, such as dimeric lipocalin muteins, also known as "duocalins". In some embodiments, the receptor binding reagent may have a single second binding site, i.e. it may be monovalent. Examples of monovalent receptor-binding reagents include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules.

Further examples of suitable proteinaceous binding molecules include EGF-like domains, Kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, Gla domains, SRCR domains, Kunitz/bovine pancreatic trypsin inhibitor domains, tendamistat, Kazal-type serine protease inhibitor domains, trefoil (P-type) domains, von Willebrand factor type C domains, anaphylatoxin-like domains, CUB domains, thyroglobulin type I lipids, , LDL receptor class A domain, sushi domain, link domain, thrombospondin type I domain, immunoglobulin domain or immunoglobulin-like domain (e.g. domain antibody or camel heavy chain antibody), C-type lectin domain, MAM domain, von Willebrand factor type A domain, somatomedin B domain, WAP type 4 disulfide core domain, F5/8 type C domain, hemopexin domain, SH2 domain, SH3 domain, laminin type EGF-like domain, C2 domain, "kappa body" (Ill.et al., Protein Eng (1997) 10, 949-57, the so-called "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), diabodies (Holliger et al., PNAS USA (1993) 90, 6444-6448), the so-called "Janusis" (Traunecker et al., EMBO J (1991) 10, 3655-3659 or Traunecker et al., Int J Cancer (1992) Suppl 7, 51-52), nanobodies, microbodies, affilins, affibodies, knottins, ubiquitins, zinc finger proteins, autofluorescent proteins or leucine-rich repeat proteins. An example of a nucleic acid molecule with antibody-like functions is the aptamer. Aptamers fold into well-defined three-dimensional motifs and show high affinity for a given target structure.

In certain aspects, the selection agent contains a binding partner C. In some aspects, the binding partner C contained in the selection agent can be, for example, hydrocarbon-based (including polymeric) and can contain nitrogen, phosphorus, sulfur, carbene, halogen or pseudohalogen groups. It can be an alcohol, an organic acid, an inorganic acid, an amine, a phosphine, a thiol, a disulfide, an alkane, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a sugar, an oligosaccharide or a polysaccharide. As a further example, it can be a cation, an anion, a polycation, a polyanion, a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube or a carbon nanofoam. In general, such a binding partner has a higher affinity for the binding site of the selection agent or the multimerization agent than other entities. Examples of respective binding partners include, but are not limited to, crown ethers, immunoglobulins, fragments thereof and proteinaceous binding molecules with antibody-like functions.

In some embodiments, the binding partner C contained in the selection substance comprises biotin, and the selection reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, the binding partner C contained in the selection substance comprises a biotin analog that reversibly binds to streptavidin or avidin, and the selection reagent comprises a streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective biotin analog. In some embodiments, the binding partner C contained in the selection substance comprises a streptavidin-binding peptide or an avidin-binding peptide, and the selection reagent comprises a streptavidin, avidin, a streptavidin analog or an avidin analog that reversibly binds to the respective streptavidin-binding peptide or avidin-binding peptide.

In some embodiments, the binding partner included in the selection agent may comprise a streptavidin binding peptide. In some embodiments, the peptide sequence comprises a sequence of the general formula His-Pro-Xaa, where Xaa is glutamine, asparagine, or methionine, e.g., the sequence shown in SEQ ID NO:9. In some embodiments, the peptide sequence has the general formula shown in SEQ ID NO:11, e.g., the general formula shown in SEQ ID NO:12. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also known as Strep-tag® and shown in SEQ ID NO:7). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II and shown in SEQ ID NO:8), which is described, for example, in U.S. Pat. No. 6,103,493 and is commercially available under the trademark Strep-Tactin®. The streptavidin-binding peptide may be, for example, a single peptide, such as the "Strep-tag®" described in U.S. Pat. No. 5,506,121, or may be a streptavidin-binding peptide in which two or more individual binding modules are arranged in sequence, such as described, for example, in WO 02/077018 or U.S. Pat. No. 7,981,632.

In some embodiments, the binding partner C of the selection agent comprises a moiety known to those skilled in the art as an affinity tag. In such embodiments, the selection reagent comprises a corresponding binding partner, e.g., an antibody or antibody fragment, known to bind to the affinity tag. Specific examples of known affinity tags include dinitrophenol or digoxigenin, oligohistidine, polyhistidine, immunoglobulin domains, maltose binding protein, glutathione-S-transferase (GST), chitin binding protein (CBP) or thioredoxin, calmodulin binding peptide (CBP), FLAG'-peptide, HA tag, VSV-G tag, HSV tag, T7 epitope, maltose binding protein (MBP), HSV epitope of sequence of herpes simplex virus glycoprotein D, "myc" epitope of transcription factor c-myc of sequence, V5 tag or glutathione-S-transferase (GST). In such embodiments, the complex formed between one or more binding sites of the selection reagent, in this case an antibody or antibody fragment, and the antigen can be competitively disrupted by adding free antigen, i.e., a free peptide (epitope tag) or a free protein (such as MBP or CBP). The affinity tag can be an oligonucleotide tag. Such an oligonucleotide tag can be used, for example, to hybridize to an oligonucleotide with a complementary sequence linked to or contained in the selection reagent.

In line with co-pending international patent application PCT/EP2012/063969 published as WO 2013/011011 (the entire contents of which are incorporated herein by reference for all purposes), the strength of the binding between the selection agent and the selection marker on the target cell may not be critical to the reversibility of the binding of the target cell to the selection agent via the selection agent. Rather, regardless of the strength of the binding, i.e., whether the dissociation constant (K _D ) for the binding between the selection agent and the selection marker via binding site B is a low affinity one, with K _D in the range of, for example, about 10 ⁻³ to about 10 ⁻⁷ M, or a high affinity one, with K _D in the range of, for example, about 10 ⁻⁷ to about 1×10 ⁻¹⁰ M, the target cell may be reversibly stained as long as the dissociation of the binding of the selection agent to the receptor molecule via binding site B occurs sufficiently fast. In this regard, the dissociation rate constant (k _off ) for the binding between the selection agent and the selection substance via binding site B may have a value of about 3×10 ⁻⁵ sec ⁻¹ or more (this dissociation rate constant is a constant that characterizes the dissociation reaction of the complex formed between binding site B of the selection agent and the selection marker on the surface of the target cell). The association rate constant (k _on ) for the association reaction between binding site B of the selection agent and the selection marker on the surface of the target cell may have any value. To ensure sufficiently reversible binding between the selection marker and the selection substance, it is advantageous to select the k _off value of the binding equilibrium to have a value of about 3×10 ^-5 sec ^-1 or more, about 5×10 ^-5 sec ^-1 or more, for example 1×10 ^-4 sec ^-1 or more, 5× ^{10 -4 sec -1} or more, 1×10 ^-3 sec ^-1 or more, 5×10 ^-3 sec ^-1 or more, ¹ ×10 ^-2 sec ^-1 or more, 1×10 ^-1 sec ^-1 or more or about 1×10 ^-4 sec ^-1 or more, 5×10 ^-4 sec ^-1 or more, 1×10 ^-3 sec ^-1 or more, 5×10 ^-3 sec ^-1 or more, 1×10 ^-2 sec ^-1 or more, 1×10 ^-1 sec ^-1 or more or 5×10 ^-1 sec ^-1 or more. It should be noted that the kinetic and thermodynamic constant values used herein refer to conditions of atmospheric pressure, i.e. 1.013 bar, and room temperature, i.e. 25°C.

In some embodiments, the selection agent has a single (monovalent) binding site B capable of specifically binding to the selection marker. In some embodiments, the selection agent has at least two (i.e. a plurality of binding sites, including three, four, or five identical binding sites B) capable of binding to the selection marker. In any of these embodiments, the binding of the selection marker via (each of) the binding sites B may have a k _off value of about 3×10 ⁻⁵ sec ⁻¹ or more. Thus, the selection agent may be a monovalent (e.g. a monovalent antibody fragment or a monovalent artificial binding molecule (proteinaceous or otherwise), such as a mutein based on a polypeptide of the lipocalin family (also known as "Anticalin®"), or a bivalent molecule, such as an antibody or a fragment in which both binding sites are retained, such as an F(ab') ₂ fragment. In some embodiments, the selection marker may be a multivalent molecule, such as a pentameric IgE molecule, so long as the k _off rate is 3×10 ⁻⁵ sec ⁻¹ or more.

In some embodiments of the present invention, it is not the k off rate (greater than or equal to 3×10 ⁻⁵ sec ⁻¹ ) of the binding of the selection agent to the selection marker on the target cell via at least one binding site B at the molecular level that allows for the (traceless) isolation of biological materials by the _reversible cell affinity chromatography techniques described herein. Rather, and as described, for example, in US Pat. No. 7,776,562 or in International Patent Application WO 02/054065, it is the low affinity binding between the selection marker and the binding site B of the selection agent, coupled with the avidity effect mediated by the immobilized selection reagent, that allows for the reversible and traceless isolation of the target cell. In these embodiments, the formation of a complex between two or more binding sites Z of the selection reagent and at least two binding partners C of the selection agent is possible, allowing for the reversible immobilization of the target cell to the affinity chromatography matrix. As described above, such low binding affinity may be characterized by a dissociation constant (K D ) in the range of about 1.0×10 ⁻³ M to about 1.0×10 ⁻⁷ M for the binding of the selection substance to the selection marker on the target cell surface via binding site _B.

In some embodiments, the selection marker may be CD4 and the selection agent specifically binds to CD4. In some aspects, the selection agent that specifically binds to CD4 may be selected from the group consisting of an anti-CD4 antibody, a bivalent antibody fragment of an anti-CD4 antibody, a monovalent antibody fragment of an anti-CD4 antibody, and a proteinaceous CD4 binding molecule with antibody-like binding properties. In some embodiments, the anti-CD4 antibody, e.g., a bivalent or monovalent antibody fragment (e.g., a CD4 Fab fragment), can be derived from the antibody 13B8.2 or a functionally active variant of 13B8.2 that retains specific binding to CD4. For example, exemplary variants of the antibody 13B8.2 or m13B8.2 are described in U.S. Pat. No. 7,482,000, U.S. Patent Application Publication No. 2014/0295458, or International Patent Application WO2013/124474, and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment, designated "m13B8.2", has the variable domains of the CD4-binding murine antibody 13B8.2 and a constant domain containing a constant human CH1 domain of gamma type for the heavy chain and a constant human light chain domain of kappa type, as described in US Patent No. 7,482,000. In some embodiments, the anti-CD4 antibody, e.g., a mutant of antibody 13B8.2, contains the amino acid substitution H91A in the variable light chain, the amino acid substitution Y92A in the variable light chain, the amino acid substitution H35A in the variable heavy chain, and/or the amino acid substitution R53A in the variable heavy chain, each according to the Kabat numbering. In some aspects, the His residue at position 91 of the light chain (position 93 in SEQ ID NO:30) is mutated to Ala and the Arg residue at position 53 of the heavy chain (position 55 in SEQ ID NO:29) is mutated to Ala, as compared to the variable domain of the 13B8.2 Fab fragment in m13B8.2. In some embodiments, the reagent that reversibly binds anti-CD4 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8000-206 or 6-8000-205 or 6-8002-100, IBA GmbH, Gottingen, Germany). In some embodiments, the selection agent comprises an anti-CD4 Fab fragment. In some embodiments, the anti-CD4 Fab fragment comprises a variable heavy chain having a sequence set forth in SEQ ID NO:29 and a variable light chain having a sequence set forth in SEQ ID NO:30. In some embodiments, the anti-CD4 Fab fragment comprises the CDRs of the variable heavy chain having a sequence set forth in SEQ ID NO:29 and the CDRs of the variable light chain having a sequence set forth in SEQ ID NO:30.

In some embodiments, the selection marker may be CD8 and the selection agent specifically binds to CD8. In some aspects, the selection agent that specifically binds to CD8 may be selected from the group consisting of an anti-CD8 antibody, a bivalent antibody fragment of an anti-CD8 antibody, a monovalent antibody fragment of an anti-CD8 antibody, and a proteinaceous CD8 binding molecule with antibody-like binding properties. In some embodiments, the anti-CD8 antibody, e.g., a bivalent or monovalent antibody fragment (e.g., a CD8 Fab fragment), can be derived from the antibody OKT8 (e.g., ATCC CRL-8014) or a functionally active variant thereof that retains specific binding to CD8. In some embodiments, the reagent that reversibly binds to anti-CD8 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog number 6-8003 or 6-8000-201, IBA GmbH, Gottingen, Germany). In some embodiments, the selection agent comprises an anti-CD8 Fab fragment. In some embodiments, the anti-CD8 Fab fragment comprises a variable heavy chain having a sequence set forth in SEQ ID NO:36 and a variable light chain having a sequence set forth in SEQ ID NO:37. In some embodiments, the anti-CD8 Fab fragment comprises the CDRs of the variable heavy chain having a sequence set forth in SEQ ID NO:36 and the CDRs of the variable light chain having a sequence set forth in SEQ ID NO:37.

In some embodiments, the selection marker may be CD3 and the selection agent specifically binds to CD3. In some aspects, the selection agent that specifically binds to CD3 may be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3 binding molecule with antibody-like binding properties. In some embodiments, the anti-CD3 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD3 Fab fragment), can be derived from the antibody OKT3 (e.g., ATCC CRL-8001; see, e.g., Stemberger et al. PLoS One. 2012; 7(4):e35798) or a functionally active variant thereof that retains specific binding to CD3. In some embodiments, the reagent that reversibly binds to anti-CD3 or a fragment thereof is commercially available or derived from a commercially available reagent (e.g., catalog no. 6-8000-201, 6-8001-100, IBA GmbH, Gottingen, Germany). In some embodiments, the selection agent comprises an anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment comprises a variable heavy chain having a sequence shown in SEQ ID NO:31 and a variable light chain having a sequence shown in SEQ ID NO:32. In some embodiments, the anti-CD3 Fab fragment comprises the CDRs of the variable heavy chain having a sequence shown in SEQ ID NO:31 and the CDRs of the variable light chain having a sequence shown in SEQ ID NO:32.

In any of the above examples, the bivalent antibody fragment may be a (Fab)2' fragment or a bivalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment and a single chain Fv fragment (scFv). In any of the above examples, the proteinaceous binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystal scaffold-based protein, an adnectin and an avimer.

C. On-column stimulation In the methods provided herein, cell selection by column chromatography step is integrated with stimulation. Thus, in certain aspects, stimulation is performed when cells are immobilized (e.g., by a selection agent) on the column during at least a portion of the selection step. In some embodiments where more than one column is used for selection, stimulation is performed on each column. In some embodiments where more than one column is used for selection, stimulation is performed on fewer columns than the total number of columns. In some embodiments where more than one column is used for selection, stimulation is performed on at least one column. In some embodiments where parallel selection is used, stimulation is performed on each column. In some embodiments where parallel selection is used, stimulation is performed on at least one column. In some embodiments where sequential selection is used, stimulation is performed on each column. In some embodiments where sequential selection is used, stimulation is performed on at least one column. In some embodiments, the stimulation conditions include stimulating or activating conditions and can deliver a stimulatory signal, e.g., a signal generated from a TCR and/or a costimulatory molecule, in cells, e.g., CD3+, CD4+, or CD8+ T cells. In some embodiments, the stimulating conditions are or include incubation of target cells (e.g., T cells) immobilized on a chromatographic matrix (e.g., stationary phase) with a stimulatory agent, such as an agent that delivers or has the ability to deliver a stimulatory signal, thereby stimulating the selected cells, or with a stimulatory reagent that includes a stimulatory agent, such as an oligomeric stimulatory reagent. In some embodiments, the stimulatory agent binds to and/or activates TCR and/or costimulatory molecules. In particular embodiments, the stimulatory reagent is an oligomeric stimulatory reagent provided herein, for example, as described in section IC-1a. In certain embodiments, stimulating a population of cells under stimulatory conditions produces or creates a population of selected stimulated cells (also referred to herein as a stimulated cell population). The selected stimulated population of cells may also be referred to herein as an output population of stimulated selected cells. In some cases, the selected stimulated population of cells may serve as an input population for downstream processing, for example, for genetic engineering as described in section IE.

In certain embodiments, the cells of the sample are selected and stimulated prior to introducing the heterologous or recombinant polynucleotide into the cells, such as by the methods, processes, or techniques described herein, e.g., in Sections I-E. In some embodiments, the output population of selected and stimulated cells is engineered to express a heterologous or recombinant protein (e.g., a chimeric antigen receptor).

In some embodiments, stimulation is considered to be initiated when a cell of the population is first stimulated or first exposed to conditions that activate or stimulate the cells and/or conditions that can activate or stimulate signals in the cells, such as signals generated from a TCR and/or a co-receptor or costimulatory molecule. In some embodiments, stimulation is initiated when the cells are first contacted or exposed to a stimulatory agent or a stimulating reagent containing a stimulatory agent, e.g., as described in Sections I-C-1a and/or II-A herein, e.g., as described in Sections II-A and/or I-C-1b herein. In certain aspects, the initiation of stimulation (also referred to herein as the initiation of incubation) occurs when target cells (e.g., T cells) of a sample immobilized on a chromatographic matrix (e.g., a stationary phase) are first contacted or exposed to a stimulatory agent or a stimulating reagent containing a stimulatory agent, e.g., an oligomeric stimulating reagent, as described in Section I-C-1b below. In some embodiments, the cells are allowed to permeate the column for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes prior to addition of the stimulating reagent (e.g., oligomeric stimulating reagent) or stimulating substance. In some embodiments, the column is washed at least one (1, 2, 3, 4, 5) time prior to addition of the stimulating reagent (e.g., oligomeric stimulating reagent) or stimulating substance. In some embodiments, the column is washed at least two times prior to addition of the stimulating substance or stimulating reagent containing the stimulating substance (e.g., oligomeric stimulating reagent).

In some embodiments, the stimulant or a stimulating reagent (e.g., an oligomeric stimulating reagent) containing the stimulant is added 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes after addition of the sample to the chromatography column (e.g., the stationary phase), or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes after addition of the sample to the chromatography column (e.g., the stationary phase). In some embodiments, the stimulant or a stimulating reagent (e.g., an oligomeric stimulating reagent) comprising the stimulant is added at, about, or at least 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or 120 minutes after adding the sample to the chromatography column (e.g., the stationary phase). In some embodiments, the stimulant or the stimulating reagent including the stimulant (e.g., oligomer stimulating reagent) is added 30, 35, 40, 45, 50, 55, or 60 minutes, or about, or at least 30, 35, 40, 45, 50, 55, or 60 minutes, after the sample is added to the chromatographic column (e.g., stationary phase). In some embodiments, the stimulant or the stimulating reagent including the stimulant (e.g., oligomer stimulating reagent) is added between about 15 and about 120 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulant or the stimulating reagent including the stimulant (e.g., oligomer stimulating reagent) is added between about 15 and about 100 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulant or the stimulating reagent including the stimulant (e.g., oligomer stimulating reagent) is added between about 15 and about 90 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 15 and about 80 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 15 and about 70 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 15 and about 60 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 15 and about 50 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 15 and about 40 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 15 and about 30 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 30 and about 120 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 30 and about 100 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 30 and about 90 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulator or the stimulating reagent comprising the stimulator (e.g., oligomer stimulating reagent) is added between about 30 and about 80 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulant or the stimulating reagent containing the stimulant (e.g., oligomer stimulating reagent) is added between about 30 and about 70 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulant or the stimulating reagent containing the stimulant (e.g., oligomer stimulating reagent) is added between about 30 and about 60 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulant or the stimulating reagent containing the stimulant (e.g., oligomer stimulating reagent) is added between about 30 and about 50 minutes, inclusive, after the sample is added to the column. In some embodiments, the stimulant or the stimulating reagent containing the stimulant (e.g., oligomer stimulating reagent) is added between about 30 and about 40 minutes, inclusive, after the sample is added to the column. In some embodiments, at least one wash step is performed before adding the stimulant or the reagent containing the stimulant (e.g., oligomer stimulating reagent) to the column.

In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulating conditions, is carried out for 1 day, about 1 day, or less than 1 day. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulating conditions, is carried out for 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours, or for less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours. In some embodiments, the stimulation, e.g., incubating the selected cells under stimulatory conditions, is performed using a method comprising the steps of: 2-24, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 hours. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulation conditions, is performed for 24 hours, about 24 hours, or less than 24 hours. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulation conditions, is performed for 12 hours, about 12 hours, or less than 12 hours. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulation conditions, is performed for 5 hours, about 5 hours, or less than 5 hours. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulation conditions, is performed for 4.5 hours, about 4.5 hours, or less than 4.5 hours. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulation conditions, is performed for 4 hours, about 4 hours, or less than 4 hours. In some embodiments, the stimulation, e.g., incubating the fixed cells under stimulation conditions, is performed for 2 hours, about 2 hours, or less than 2 hours.

In certain embodiments, 50×10 ⁶ , 100×10 ⁶ , 150×10 ⁶ , 200×10 ⁶ , 250×10 ⁶ , 300×10 ⁶ , 350×10 ⁶ , 400×10 ⁶ , 450×10 ⁶ , 500×10 ⁶ , 550×10 ⁶ , 600×10 ⁶ , 700×10 ^{6 , 800×10 6 , 900×10 6 , 1,000×10 6 , 1250×10 6 , 1500×10 6 , 1750 × 10 6 ,} 2000×10 ⁶ , 2250×10 ⁶ , 2500×10 ⁶ , 2750×10 ⁶ , 3000×10 ⁶ 3250×10 ⁶ , 3500 10 ⁶ , 3750×10 ⁶ , 4000×10 ⁶ , 4250×10 ⁶ , 4500×10 ⁶ , 4750×10 ⁶ or 5000×10 ⁶ pieces, about 50×10 ⁶ , 100×10 ⁶ , 150×10 ⁶ , 200×10 ⁶ , 250×10 ⁶ , 300×10 ⁶ , 350×10 ⁶ , 400×10 ⁶ , 450×10 ⁶ , 500×10 ⁶ , 550×10 ⁶ , 600×10 ⁶ , 700×10 ⁶ , 800×10 ⁶ , 900×10 ⁶ , 1,000× ¹⁰⁶ , 1250× ¹⁰⁶ , 1500× ¹⁰⁶ , 1750× ¹⁰⁶ , 2000× ¹⁰⁶ , 2250× ¹⁰⁶ , 2500× ¹⁰⁶ , 2750× ¹⁰⁶ , 3000×106, 3250× ¹⁰⁶ , 3500× ^{106 ,} 3750× ¹⁰⁶ , 4000×106, 4250× ¹⁰⁶ , 4500× ¹⁰⁶ , 4750× ¹⁰⁶ or 5000× ¹⁰⁶ , or at least 50× ¹⁰⁶ , 100× ¹⁰⁶ , 150× ¹⁰⁶ , 200× ¹⁰⁶ , 250 ^{× 106} , 300×10 ⁶ , 350×10 ⁶ , 400×10 ⁶ , 450×10 ⁶ , 500×10 ⁶ , 550×10 ⁶ , 600×10 ⁶ , 700×10 ⁶ , 800×10 ⁶ , 900×10 ⁶ , 1,000×10 ⁶ , 1250×10 ⁶ , 1500×10 ⁶ , 1750×10 ⁶ , 2000×10 ⁶ , 2250×10 ⁶ , 2500×10 ⁶ , 2750×10 ⁶ , 3000×10 ⁶ 3250×10 ⁶ , 3500 10 ⁶ , 3750×10 ⁶ , 4000×10 ⁶ , 4250×10 ⁶ , 4500×10 ⁶ , 4750×10 ⁶ or 5000×10 ⁶ cells, or any number of cells between any of the above, are stimulated (e.g., incubated under stimulating conditions). In some embodiments, the selected cells are immobilized on a single column (e.g., containing a chromatography matrix). For example, the entire amount of selected cells from a sample is immobilized on a single column, and the immobilized cells on the single column are incubated under stimulating conditions. In some embodiments, the selected cells are immobilized on two columns (e.g., each containing a chromatography matrix). For example, the entire amount of selected cells from a sample is immobilized on two columns (e.g., each column (e.g., chromatography matrix) contains half or about half of the total amount of cells immobilized thereon), and the immobilized cells on the two columns are incubated under stimulating conditions. In certain embodiments, the cells on the chromatography matrix (e.g., stationary phase), e.g., the selected cells (e.g., T cells), are at a concentration of 0.01×10 ⁶ cells/mL, ^0.1 ×10 ⁶ cells/mL, 0.5×10 ⁶ cells/mL ^{, 1.0} ×10 ⁶ cells/mL, 1.5×10 ⁶ cells/mL, 2.0×10 6 cells/mL, 2.5×10 ⁶ cells/mL, 3.0×10 6 cells/ ^mL , 4.0×10 ⁶ cells/mL, 5.0×10 ⁶ cells/mL, 10×10 6 cells/mL, 50×10 ⁶ cells/mL, 75×10 ⁶ cells/mL, 100×10 ⁶ cells/mL, 125×10 6 cells/mL, 150×10 ⁶ cells/mL, or 200×10 ⁶ cells/mL, or at a concentration of about 0.01×10 ⁶ cells/ ^{mL , 0.1 x 106} cells/ ^mL , ^0.5 x 106 cells/mL, ^1.0 x 106 cells/mL ^, 1.5 x 106 cells/mL, 2.0 x ¹⁰⁶ cells/mL, 2.5 x ¹⁰⁶ cells/mL, 3.0 x 106 cells/mL, 4.0 x ¹⁰⁶ cells/mL, 5.0 x 106 cells/mL, 10 x ¹⁰⁶ cells/mL, 50 x 106 cells/mL, 75 x 106 cells/mL, 100 x ¹⁰⁶ cells/mL, 125 x ¹⁰⁶ cells/mL, 150 x ¹⁰⁶ cells/mL or 200 x ¹⁰⁶ cells/mL or at least 0.01 x 106 cells/mL, 0.1 x ¹⁰⁶ cells/mL, ^{0.5 x 106 cells} /mL, 1.0 x 10 The cells are ^stimulated (e.g. ^, incubated under stimulatory conditions, such as ⁱⁿ the presence ^of a stimulatory agent) at a ^{density of 1.5} × ¹⁰⁶ cells/mL, ^2.0 × ¹⁰⁶ cells/mL, 2.5× ¹⁰⁶ cells/mL, 3.0×106 cells/mL, 4.0× ¹⁰⁶ cells/mL, 5.0×106 cells/mL, 10× ¹⁰⁶ cells/mL, 50×106 cells/mL, 75× ^{106 cells/mL, 100×106} cells/mL, 125×106 cells/mL, 150×106 cells/mL or 200× ¹⁰⁶ cells/mL. In certain embodiments, the immobilized cells on the stationary phase, such as selected cells (e.g., T cells), are stimulated or subjected to stimulation at a density of 100±25 million cells/mL or about 100±25 million cells/mL, and incubated under stimulating conditions, such as in the presence of a stimulating substance. In certain embodiments, the immobilized cells on the stationary phase, such as selected cells (e.g., T cells), are stimulated or subjected to stimulation at a density of 3.0× ¹⁰⁶ cells/mL, about 3.0× ¹⁰⁶ cells/mL, or at least 3.0× ¹⁰⁶ cells/mL, and incubated under stimulating conditions, such as in the presence of a stimulating substance. In certain embodiments, the selected cells are viable cells.

In some embodiments, the stimulatory agent or stimulatory reagent comprising the stimulatory agent is added to the column at a concentration of, or about, or at least, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3 μg per 1×10 ⁶ cells. In some embodiments, the stimulator or stimulating reagent comprising the stimulator is added to the column containing the fixed cells at a concentration of, or about, or at least, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25 μg per 1×10 ⁶ cells. In some embodiments, the stimulator or stimulating reagent comprising the stimulator is added to the column at a concentration of, or about, 1-2 μg per 1×10 ⁶ cells. In some embodiments, the stimulating reagent is an oligomeric stimulating reagent. In some embodiments, the oligomeric stimulating reagent is added to the column containing the fixed cells at a concentration of, or about, 1-2 μg per 1×10 ⁶ cells. In some embodiments, 5×10 ⁸ oligomeric stimulation reagent is added to the column containing the immobilized cells. If more than one column contains immobilized cells for stimulation, a concentration or amount of stimulant or stimulation reagent containing a stimulant (e.g., oligomeric stimulation reagent) described herein is added or applied to each column.

In some embodiments, the conditions for stimulation can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some embodiments, the temperature is at or about 37°C. In some embodiments, the oxygen content and carbon dioxide content are managed using gas exchange.

In certain embodiments, the stimulatory conditions include incubating the cells, e.g., selected cells of the sample, with and/or in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind and/or are capable of binding to a receptor expressed by and/or endogenous to the selected cell (e.g., T cell). In certain embodiments, the one or more cytokines are or include members of the four-alpha-helical bundle family of cytokines. In some embodiments, members of the four-alpha-helical bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include IL-2.

In certain embodiments, the amount or concentration of one or more cytokines is measured and/or quantified using International Units (IU). International Units may be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar biologically active substances. In some embodiments, IU is or includes a unit of measurement of the potency of a biological preparation by comparison to an international reference standard of a particular weight and strength, e.g., WHO 1st International Standard for Human IL-2, 86/504. International Units are the only recognized standardized method for reporting biological activity units that are published and obtained from international collaborative research efforts. In certain embodiments, IU for a population, sample, or source of cytokines may be obtained by product comparison testing with a similar WHO standard. For example, in some embodiments, the IU/mg of a population, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to a WHO standard IL-2 product (NIBSC code: 86/500), a WHO standard IL-17 product (NIBSC code: 90/530), and a WHO standard IL-15 product (NIBSC code: 95/554), respectively.

In some embodiments, biological activity in IU/mg is equal to (ED50 in ng/ml)-1×106. In certain embodiments, the ED50 of recombinant human IL-2 or IL-15 is equal to the concentration required for half-maximal stimulation of cell proliferation (XTT cleavage) by CTLL-2 cells. In certain embodiments, the ED50 of recombinant human IL-7 is equal to the concentration required for half-maximal stimulation for proliferation of PHA-activated human peripheral blood lymphocytes. Details regarding assays and calculations of IU for IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379 (1-2): 1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114 (1-2): 3-9; details regarding assays and calculations of IU for IL-15 are discussed in Soman et al. Journal of Immunological Methods (2009) 348 (1-2): 83-94.

In some embodiments, cells, e.g., selected cells of a sample, are stimulated or subjected to stimulation in the presence of a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 IU/mL and 1,000 IU/mL, between 10 IU/mL and 50 IU/mL, between 50 IU/mL and 100 IU/mL, between 100 IU/mL and 200 IU/mL, between 100 IU/mL and 500 IU/mL, between 250 IU/mL and 500 IU/mL, or between 500 IU/mL and 1,000 IU/mL.

In some embodiments, cells, e.g., selected cells of a sample, are stimulated or subjected to stimulation in the presence of recombinant IL-2, e.g., human recombinant IL-2, at a concentration of 1 IU/mL to 500 IU/mL, 10 IU/mL to 250 IU/mL, 50 IU/mL to 200 IU/mL, 50 IU/mL to 150 IU/mL, 75 IU/mL to 125 IU/mL, 100 IU/mL to 200 IU/mL or 10 IU/mL to 100 IU/mL. In certain embodiments, cells, e.g., selected cells of a sample, have an IL-1 concentration of 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 100 IU/mL, or about The cells are stimulated or subjected to stimulation in the presence of recombinant IL-2 at a concentration of 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 100 IU/mL. In some embodiments, the cells, e.g., selected cells of the sample, are stimulated or subjected to stimulation in the presence of 100 IU/mL or about 100 IU/mL of recombinant IL-2, e.g., human recombinant IL-2.

In some embodiments, cells, e.g., selected cells of a sample, are stimulated or subjected to stimulation in the presence of recombinant IL-7, e.g., human recombinant IL-7, at a concentration of 100 IU/mL to 2,000 IU/mL, 500 IU/mL to 1,000 IU/mL, 100 IU/mL to 500 IU/mL, 500 IU/mL to 750 IU/mL, 750 IU/mL to 1,000 IU/mL or 550 IU/mL to 650 IU/mL. In certain embodiments, the cells, e.g., input cells, have a concentration of 50 IU/mL, 100 IU/mL, 150 IU/mL, 200 IU/mL, 250 IU/mL, 300 IU/mL, 350 IU/mL, 400 IU/mL, 450 IU/mL, 500 IU/mL, 550 IU/mL, 600 IU/mL, 650 IU/mL, 700 IU/mL, 750 IU/mL, 800 IU/mL, 750 IU/mL, 750 IU/mL, 750 IU/mL, or 1,000 IU/mL, or about The cells are stimulated or subjected to stimulation in the presence of IL-7 at a concentration of 50 IU/mL, 100 IU/mL, 150 IU/mL, 200 IU/mL, 250 IU/mL, 300 IU/mL, 350 IU/mL, 400 IU/mL, 450 IU/mL, 500 IU/mL, 550 IU/mL, 600 IU/mL, 650 IU/mL, 700 IU/mL, 750 IU/mL, 800 IU/mL, 750 IU/mL, 750 IU/mL, 750 IU/mL, or 1,000 IU/mL. In certain embodiments, the cells, e.g., selected cells of the sample, are stimulated or subjected to stimulation in the presence of 600 IU/mL or about 600 IU/mL of recombinant IL-7, e.g., human recombinant IL-7.

In some embodiments, cells, e.g., selected cells of a sample, are stimulated or subjected to stimulation in the presence of recombinant IL-15, e.g., human recombinant IL-15, at a concentration of 1 IU/mL to 500 IU/mL, 10 IU/mL to 250 IU/mL, 50 IU/mL to 200 IU/mL, 50 IU/mL to 150 IU/mL, 75 IU/mL to 125 IU/mL, 100 IU/mL to 200 IU/mL or 10 IU/mL to 100 IU/mL. In certain embodiments, the cells, e.g., the cells of the input population, are administered 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 200 IU/mL, or about 5 The cells are stimulated or subjected to stimulation in the presence of recombinant IL-15 at a concentration of 0 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 200 IU/mL. In some embodiments, the cells, e.g., selected cells of the sample, are stimulated or subjected to stimulation in the presence of 100 IU/mL or about 100 IU/mL of recombinant IL-15, e.g., human recombinant IL-15.

In particular embodiments, the cells, e.g., selected cells of the sample, are stimulated or subjected to stimulation under stimulatory conditions in the presence of IL-2, IL-7 and/or IL-15. In some embodiments, the IL-2, IL-7 and/or IL-15 are recombinant IL-2, IL-7 and/or IL-15. In certain embodiments, the IL-2, IL-7 and/or IL-15 are human IL-2, IL-7 and/or IL-15. In particular embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7 and/or IL-15. In certain embodiments, the cells, e.g., selected cells of the sample, are stimulated or subjected to stimulation under stimulatory conditions in the presence of recombinant IL-2, IL-7 and IL-15. In certain embodiments, the cells are stimulated or subjected to stimulation under stimulatory conditions in the presence of 100 IU/mL or about 100 IU/mL recombinant IL-2, 600 IU/mL or about 600 IU/mL recombinant IL-7, and 100 IU/mL or about 100 IU/mL recombinant IL-15. In some embodiments, the stimulatory conditions further include glutamine.

The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and/or stimuli such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

In some aspects, stimulation is performed according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother., 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the stimulation is performed in serum-free medium. In some embodiments, the serum-free medium is a defined cell culture medium and/or a chemically defined cell culture medium. In certain embodiments, the serum-free medium is a restricted culture medium that has been processed, e.g., filtered to remove inhibitors and/or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins, and/or attachment factors.

In some embodiments, stimulation is performed in serum-free medium as described in Section III herein or PCT/US2018/064627. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T Cell Expansion Basal Medium (ThermoFisher). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion and/or activation of cells (e.g., T cells), such as those provided by additional supplements (e.g., OpTmizer™ T Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium comprises a serum replacement supplement, such as immune cell serum replacement, e.g., ThermoFisher, #A2596101, CTS™ immune cell serum replacement, or Smith et al. Clin Transl Immunology. 2015 Jan;4(1):e31. In some embodiments, the serum-free medium further comprises a free amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15.

In some embodiments, the stimulation, e.g., incubation under stimulating conditions, is performed at room temperature (e.g., at or about 23°C). In some embodiments, the stimulation, e.g., incubation under stimulating conditions, is performed at or about 37°C.

The methods provided herein allow for the collection or elution of selected cells from a chromatography column without the addition of a competitor or free binder to elute the cells from the stationary phase. In some embodiments, the detachment of the selected cells from the column is accomplished by on-column stimulation. In some embodiments, the detached cells may remain bound to the stimulant or the stimulating reagent containing the stimulant, e.g., if the stimulant or the stimulating reagent containing the stimulant is not bound, e.g., directly or indirectly, to the stationary phase of the chromatography column. Thus, in some embodiments, the detached cells may remain under stimulating conditions after detachment from the column and/or when collected and/or eluted. In some embodiments, the stimulating conditions are maintained for a period of time after removal (e.g., collection or elution) from the column. In some embodiments, at least a portion of the stimulation in the presence of the stimulant or the stimulating reagent containing the stimulant is performed, e.g., under centrifugal rotation, in the internal cavity of a centrifuge chamber, as described in International Publication No. WO2016/073602.

In some embodiments, stimulation performed after collection or elution of cells from the column is generally performed under mixing conditions, e.g., under rotational motion, generally at a relatively low force or speed, e.g., a speed less than that used to pellet the cells, e.g., at a speed of 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm or 1500 rpm or 1700 rpm, or about 600 rpm, 1000 rpm or 1500 rpm or 1700 rpm, or at least 600 rpm, 1000 rpm or 1500 rpm or 1700 rpm), e.g., a speed at which the RCF at the sample or wall of the chamber or other container is about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the rotation is performed using repeat intervals of such slow rotation followed by a rest period, e.g., rotation and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, e.g., rotation for approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds. In some embodiments, the stimulation performed after collection or elution of the cells from the column is performed under mixing conditions, e.g., under rocking. In certain embodiments, the stimulation is performed under static conditions, e.g., without centrifugation, shaking, rotation, rocking, or perfusion, e.g., continuous or semi-continuous perfusion of medium.

In some embodiments, the eluted and/or collected cells, e.g., cells still bound to the stimulant or the stimulating reagent containing the stimulant, are transferred (e.g., transferred under sterile conditions) to a container such as a bag or vial and placed in an incubator. In certain embodiments, the incubator is set at, or about, or at least, 16°C, 24°C, or 35°C. In some embodiments, the incubator is set at, or about, 37°C, or 37°C ±2°C, ±1°C, ±0.5°C, or ±0.1°C. In certain embodiments, stimulation under static conditions is performed in a cell culture bag placed in an incubator. In some embodiments, stimulation under rocking conditions is performed in a cell culture bag placed in an incubator. In some embodiments, the culture bag is composed of a single-web polyolefin gas-permeable film that allows monocytes, if present, to adhere to the bag surface.

1. Use of Stimulating Substances and Stimulating Reagents for On-Column Stimulation In certain aspects, the stimulating conditions include incubating target cells (e.g., T cells) immobilized on a chromatographic matrix (e.g., stationary phase) with one or more stimulating substances. In some embodiments, the stimulating substance is included in the stimulating reagent. In some embodiments, the stimulating substance is directly or indirectly bound to the chromatographic matrix (e.g., stationary phase) of the chromatographic column. In some embodiments, the stimulating substance is indirectly bound to the chromatographic matrix (e.g., stationary phase) of the chromatographic column, e.g., via a selection reagent, e.g., as described herein in Section II-A and/or Section IB, or a stimulating reagent, e.g., as described herein in Section II-A and/or Section IC-1b. In some embodiments, the stimulating substance is included in the stimulating reagent. In some embodiments, the stimulating reagent is bound to the chromatographic matrix (e.g., stationary phase) of the chromatographic column. In some embodiments, the stimulating reagent is covalently bound to the chromatographic matrix (e.g., stationary phase). In some embodiments, the stimulating substance is non-covalently bound to the chromatographic matrix (e.g., stationary phase).

In some embodiments, the stimulating reagent is not bound to or associated with a solid support, stationary phase, bead, microparticle, magnetic particle, and/or matrix (e.g., a chromatography matrix). In some embodiments, the stimulating reagent is flexible, does not contain a metallic or magnetic core, is composed entirely or primarily of organic multimers, and/or is not rigid. In some embodiments, the stimulating reagent is soluble. In some embodiments, the stimulating reagent is an oligomeric stimulating reagent (see, e.g., Section I-C-1b). In some embodiments, the oligomeric stimulating reagent is soluble.

In certain embodiments, initiation of stimulation occurs when cells are incubated with or contacted with a stimulating agent. Thus, in some embodiments where the stimulating agent is directly or indirectly bound to the chromatographic matrix (e.g., stationary phase) of the column, e.g., via a selection or stimulating agent, initiation of stimulation occurs when a sample containing target cells is added to the chromatographic matrix (e.g., stationary phase) of the column. In some embodiments, if the stimulating agent is included in a stimulating agent that is not associated (e.g., bound) with the chromatographic matrix (e.g., stationary phase), initiation of stimulation occurs when the stimulating agent (e.g., oligomeric stimulating agent) is added to the stationary phase on which the target cells of the sample are immobilized. In some embodiments, if the stimulating agent is not directly or indirectly bound to the chromatographic matrix (e.g., stationary phase) and is not included in the stimulating agent (e.g., oligomeric stimulating agent), initiation of stimulation occurs when the stimulating agent is added to the chromatographic matrix (e.g., stationary phase).

In some embodiments, the stimulatory conditions or stimulatory reagents (e.g., oligomeric stimulatory reagents) include one or more stimuli capable of activating an intracellular signaling domain of a TCR complex. In some embodiments, the one or more stimuli are capable of activating an intracellular signaling domain of a TCR complex. In some embodiments, stimuli contemplated herein include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, or any other biomolecules that have affinity for a desired target. In some embodiments, the desired target is a T cell receptor and/or a component of a T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, e.g., CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, the stimulatory agent is an antibody or an antigen-binding fragment thereof, e.g., a Fab.

In some embodiments, the stimulatory reagent (e.g., oligomeric stimulatory reagent) contains one or more stimulatory substances that bind to one or more of the following macromolecules on a cell (e.g., a T cell): CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHCI, MHCII, CTLA-4, ICOS, including PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-gamma R, TNF-alpha R, IL-4R, IL-10R, CD18/CD11a (LFA-1), CD62L (L-selectin), CD29/CD49d (VLA-4), Notch ligands (e.g. delta-like 1/4, Jagged 1/2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7 and CXCR3 or fragments thereof, and the corresponding ligands for these macromolecules or fragments thereof. In some embodiments, the stimulatory agent specifically binds to one or more of the following macromolecules on a cell (e.g., a T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and/or CD45RO.

In some embodiments, the stimulatory agent is an antibody that binds to and/or recognizes one or more components of the T cell receptor. In particular embodiments, the stimulatory agent is an anti-CD3 antibody. In certain embodiments, the stimulatory agent is an antibody that binds to and/or recognizes a costimulatory molecule. In certain embodiments, the stimulatory agent is an anti-CD28 antibody. In some embodiments, the stimulatory reagent comprises an anti-CD28 antibody and an anti-CD3 antibody (e.g., a stimulatory agent). In some embodiments, the stimulatory reagent comprises one or more stimulatory agents. In some embodiments, the stimulatory reagent comprises a first stimulatory agent and a second stimulatory agent. In some embodiments, the first stimulatory agent is an anti-CD3 antibody or antigen-binding fragment thereof, such as those described herein, and the second stimulatory agent is an anti-CD28 antibody or antigen-binding fragment thereof, such as those described herein. In some embodiments, the first stimulatory agent is an anti-CD3 Fab, such as those described herein, and the second stimulatory agent is an anti-CD28 Fab, such as those described herein.

In some embodiments, for example when the stimulatory agent is not bound to a stimulating reagent (e.g., an oligomeric stimulating reagent) or a selection reagent, the stimulatory agent is an antibody, a bivalent antibody fragment, a F(ab) ₂ , or a bivalent single chain Fv fragment. In some embodiments, PMA/ionomycin may be used to stimulate the cells.

In some embodiments, cells, e.g., selected cells of a sample, are stimulated or subjected to stimulation at a ratio of stimulator to cells of 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1 or about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In certain embodiments, the ratio of stimulatory agent to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, between 1.1:1 and 0.9:1. In certain embodiments, the ratio of stimulatory agent to cells is about 1:1 or is 1:1. In certain embodiments, the ratio of stimulatory agent to cells is about 0.3:1 or is 0.3:1. In certain embodiments, the ratio of stimulatory agent to cells is about 0.2:1 or is 0.2:1.

In some embodiments, cells are administered at ^or about 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 1.2 μg, 1.4 μg, 1.6 μg, 1.8 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg per 10 cells. In the presence of 1.4μg, 1.6μg, 1.8μg, 2μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg or 10μg, or at least 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.05μg, 0.1μg, 0.2μg, 0.3μg, 0.4μg, 0.5μg, 0.75μg, 1μg, 1.2μg, 1.4μg, 1.6μg, 1.8μg, 2μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg or 10μg of stimulating reagent, stimulated or subjected to stimulation. In some embodiments, cells are stimulated or subjected to stimulation in the presence of 4μg or about 4μg per ¹⁰⁶ cells. In a particular embodiment, the cells are stimulated or stimulated in the presence of 3 μg or about 3 μg per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or stimulated in the presence of 2.5 μg or about 2.5 μg per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or stimulated in the presence of 2 μg or about 2 μg per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or stimulated in the presence of 1.8 μg or about 1.8 μg per 10 ^{6 cells. In a particular embodiment, the cells are stimulated or stimulated in the presence of 1.6 μg or about 1.6 μg per 10 6 cells} . In a particular embodiment, the cells are stimulated or stimulated in the presence of 1.4 μg or about 1.4 μg per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or subjected to stimulation in the presence of 1.2 μg or about 1.2 μg per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or subjected to stimulation in the presence of 1 μg or about 1 μg per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or subjected to stimulation in the presence of 0.8 μg or about 0.8 μg per 10 ⁶ cells. In various embodiments, the cells are stimulated or subjected to stimulation in the presence of 0.8 μg or about 0.8 μg per 10 ⁶ cells.

a. Stimulatory Agents As mentioned above, in certain aspects, the methods provided herein use stimulatory agents. In some embodiments, the agent described in Section II-B is a stimulatory agent. In some embodiments, the stimulatory agent binds to a molecule on the surface of a cell, and the binding of the stimulatory agent to the molecule can induce, deliver or modulate a stimulatory signal in the cell. In some examples, the cell surface molecule (e.g., a receptor) is a signaling molecule. In some such examples, the stimulatory agent has the ability to specifically bind to a signaling molecule expressed by one or more target cells (e.g., T cells). In some examples, the stimulatory agent is any agent that has the ability to induce or deliver a stimulatory signal in a cell (e.g., T cells) when bound to a cell surface molecule such as a receptor. In some embodiments, the stimulatory signal can be immunostimulatory, in which case the stimulatory agent has the ability to induce, deliver or modulate a signal involved in or stimulating an immune response by a cell (e.g., T cells), for example, increasing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other functional activities of an immune cell. In some embodiments, the stimulatory signal can be inhibitory, in which case the stimulatory agent has the ability to induce, deliver or modulate a stimulatory signal in a cell (e.g., a T cell) that is involved in or inhibits an immune response, e.g., inhibiting or reducing immune cell proliferation or expansion, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or other functional activity or activities of an immune cell.

In some embodiments, the stimulatory agent is a first stimulatory agent. In some embodiments, the first stimulatory agent binds to a receptor molecule on the surface of selected cells of the sample. Thus, optionally, the first stimulatory agent delivers, induces, or modulates a stimulatory signal. In some aspects, the delivery, induction, or modulation of a stimulatory signal by the first stimulatory agent results in stimulation of the cell. Thus, optionally, the first stimulatory agent delivers a stimulatory signal or provides a primary activation signal to the cell, thereby stimulating and/or activating the cell. In some embodiments, the first stimulatory agent further induces downregulation of a selection marker. As used herein, downregulation can include a reduction in expression, e.g., cell surface expression, of the selection marker compared to a previous time point.

In some embodiments, the target cell (e.g., a T cell) comprises a TCR/CD3 complex and a costimulatory molecule, such as CD28. In this case, the first stimulatory agent binds to the TCR/CD3 complex, thereby delivering a stimulatory signal (e.g., a primary signal, e.g., a primary activation signal) in the T cell, and the second stimulatory agent binds to the costimulatory CD28 molecule. In certain aspects, the first stimulatory agent and/or the second stimulatory agent further induce downregulation of a selection marker (e.g., a selection marker used to immobilize the target cell (e.g., a T cell)).

In some embodiments, the first stimulatory agent delivers a TCR/CD3 complex-associated stimulatory signal (e.g., a primary signal) in a cell, e.g., a T cell. In some embodiments, the first stimulatory agent specifically binds to a molecule containing an immunoreceptor tyrosine-based activation motif, or ITAM. In some aspects, the first stimulatory agent specifically binds to CD3. Optionally, the first stimulatory agent that specifically binds to CD3 can be selected from the group consisting of an anti-CD3 antibody, a bivalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3 antibody, and a proteinaceous CD3 binding molecule with antibody-like binding properties. The bivalent antibody fragment can be a F(ab') ₂ -fragment or a bivalent single-chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv). In some cases, the proteinaceous CD3 binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on an ankyrin scaffold, a protein based on a crystal scaffold, an adnectin or an avimer.

In some embodiments, the anti-CD3 Fab fragment can be derived from a CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™, see also U.S. Pat. No. 4,361,549). The heavy and light chain variable domains of the anti-CD3 antibody OKT3 are described in Arakawa et al J. Biochem. 120, 657-662 (1996) and comprise the amino acid sequences shown in SEQ ID NOs: 31 and 32, respectively. In some embodiments, the anti-CD3 Fab comprises the variable heavy and variable light chain CDRs shown in SEQ ID NOs: 31 and 32, respectively.

In some embodiments, the stimulatory agent is a second stimulatory agent. In some embodiments, the second stimulatory agent binds to a molecule on the cell surface, e.g., a cell surface molecule, e.g., a receptor molecule. In some embodiments, the second stimulatory agent has the ability to enhance, attenuate, or modify a stimulatory signal delivered via a molecule bound by the first stimulatory agent. In some embodiments, the second stimulatory agent delivers, induces, or modulates a stimulatory signal, e.g., a second or additional stimulatory signal. In some aspects, the second stimulatory agent enhances or strengthens a stimulatory signal induced by the first stimulatory agent. In some embodiments, the second stimulatory agent can bind to an accessory molecule and/or stimulate or induce a co-stimulatory signal or a secondary stimulatory signal. In some aspects, the second stimulatory agent binds to a costimulatory molecule and/or provides a costimulatory signal.

In some embodiments, the stimulatory agent, which can be a second stimulatory agent, binds, e.g., specifically binds, to a second molecule, which can be a costimulatory molecule, an accessory molecule, a cytokine receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or the TNF receptor family.

In some embodiments, the molecule on the cell, e.g., T cell, may be CD28 and the stimulator (e.g., it may be a second stimulator) specifically binds to CD28. In some aspects, the stimulator (e.g., it may be a second stimulator) specifically binds to CD28 may be selected from the group consisting of an anti-CD28 antibody, a bivalent antibody fragment of an anti-CD28 antibody, a monovalent antibody fragment of an anti-CD28 antibody, and a proteinaceous CD28 binding molecule with antibody-like binding properties. The bivalent antibody fragment may be a F(ab') ₂ -fragment or a bivalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv). The proteinaceous CD28 binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystal scaffold-based protein, an adnectin, and an avimer.

In some embodiments, the anti-CD28 Fab fragment can be derived from the antibody CD28.3 (deposited as a synthetic single chain Fv construct in GenBank Accession No. AF451974.1; see also Vanhove et al, BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570), whose variable heavy and variable light chains comprise SEQ ID NOs: 33 and 34, respectively. In some embodiments, the anti-CD28 Fab comprises the CDRs of the variable heavy and variable light chains shown in SEQ ID NOs: 33 and 34, respectively.

In some embodiments, the molecule on the cell, e.g., T cell, is CD90 and the stimulator (e.g., which can be a second stimulator) specifically binds to CD90. In some aspects, the stimulator (e.g., which can be a second stimulator) that specifically binds to CD90 can be selected from the group consisting of an anti-CD90 antibody, a bivalent antibody fragment of an anti-CD90 antibody, a monovalent antibody fragment of an anti-CD90 antibody, and a proteinaceous CD90 binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art. See, e.g., anti-CD90 antibody G7 (Biolegend, catalog number 105201).

In some embodiments, the molecule on the cell, e.g., T cell, is CD95 and the stimulator (e.g., which can be a second stimulator) specifically binds to CD95. In some aspects, the stimulator (e.g., which can be a second stimulator) that specifically binds to CD95 can be selected from the group consisting of an anti-CD95 antibody, a bivalent antibody fragment of an anti-CD95 antibody, a monovalent antibody fragment of an anti-CD95 antibody, and a proteinaceous CD95 binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art. For example, in some aspects, the anti-CD90 antibody can be monoclonal mouse anti-human CD95 CH11 (Upstate Biotechnology, Lake Placid, NY), anti-CD95 mAb 7C11, or anti-APO-1, e.g., as described in Paulsen et al. Cell Death & Differentiation 18.4 (2011):619-631.

In some embodiments, the molecule on the cell, e.g., a T cell or a B cell, can be CD137 and the stimulator (e.g., it can be a second stimulator) specifically binds to CD137. In some aspects, the stimulator (e.g., it can be a second stimulator) that specifically binds to CD137 can be selected from the group consisting of an anti-CD137 antibody, a bivalent antibody fragment of an anti-CD137 antibody, a monovalent antibody fragment of an anti-CD137 antibody, and a proteinaceous CD137 binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art. For example, the anti-CD137 antibody can be LOB12, IgG2a or LOB12.3, IgG1 as described in Taraban et al. Eur J Immunol. 2002 Dec;32(12):3617-27. See, e.g., US6569997, US6303121, and Mittler et al. Immunol Res. 2004;29(1-3):197-208.

In some embodiments, the molecule on the cell, e.g., a B cell, can be CD40 and the stimulator, e.g., the stimulator (e.g., which can be a second stimulator, e.g., a second stimulator) specifically binds to CD40. In some aspects, the stimulator (which can be a second stimulator, e.g., a second stimulator) that specifically binds to CD40 can be selected from the group consisting of an anti-CD40 antibody, a bivalent antibody fragment of an anti-CD40 antibody, a monovalent antibody fragment of an anti-CD40 antibody, and a proteinaceous CD40 binding molecule with antibody-like binding properties.

In some embodiments, the molecule on the cell, e.g., a T cell, can be CD40L (CD154) and the stimulator (e.g., which can be a second stimulator) specifically binds to CD40L. In some aspects, the stimulator (e.g., which can be a second stimulator) that specifically binds to CD40L can be selected from the group consisting of an anti-CD40L antibody, a bivalent antibody fragment of an anti-CD40L antibody, a monovalent antibody fragment of an anti-CD40L antibody, and a proteinaceous CD40L binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art. For example, the anti-CD40L antibody can in some aspects be Hu5C8 as described in Blair et al. JEM vol.191 no.4 651-660. See also, e.g., WO1999061065, US20010026932, US7547438, WO2001056603.

In some embodiments, the molecule on the cell, e.g., T cell, may be an inducible T cell costimulatory molecule (ICOS), and the stimulator (e.g., it may be a second stimulator) specifically binds to ICOS. In some aspects, the stimulator (e.g., it may be a second stimulator) that specifically binds to ICOS may be selected from the group consisting of an anti-ICOS antibody, a bivalent antibody fragment of an anti-ICOS antibody, a monovalent antibody fragment of an anti-ICOS antibody, and a proteinaceous ICOS binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be from any known in the art. See, e.g., US20080279851 and Deng et al. Hybrid Hybridomics. 2004 Jun;23(3):176-82.

In some embodiments, the molecule on the cell, e.g., a T cell, can be a Linker for Activation of T cells (LAT), and the stimulator (e.g., it can be a second stimulator) specifically binds to LAT. In some aspects, the stimulator (e.g., it can be a second stimulator) that specifically binds to LAT can be selected from the group consisting of an anti-LAT antibody, a bivalent antibody fragment of an anti-LAT antibody, a monovalent antibody fragment of an anti-LAT antibody, and a proteinaceous LAT binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art.

In some embodiments, the molecule on the cell, e.g., a T cell, can be CD27 and the stimulator (e.g., it can be a second stimulator) specifically binds to CD27. In some aspects, the stimulator (e.g., it can be a second stimulator) that specifically binds to CD27 can be selected from the group consisting of an anti-CD27 antibody, a bivalent antibody fragment of an anti-CD27 antibody, a monovalent antibody fragment of an anti-CD27 antibody, and a proteinaceous CD27 binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art. See, e.g., WO2008051424.

In some embodiments, the molecule on the cell, e.g., T cell, can be OX40 and the stimulator (e.g., it can be a second stimulator) specifically binds to OX40. In some aspects, the stimulator (e.g., it can be a second stimulator) that specifically binds to OX40 can be selected from the group consisting of an anti-OX40 antibody, a bivalent antibody fragment of an anti-OX40 antibody, a monovalent antibody fragment of an anti-OX40 antibody, and a proteinaceous OX40 binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be from any known in the art. See, e.g., WO2013038191, Melero et al. Clin Cancer Res. 2013 Mar 1;19(5):1044-53.

In some embodiments, the molecule on the cell, e.g., T cell, may be HVEM and the stimulator (e.g., it may be a second stimulator) specifically binds to HVEM. In some aspects, the stimulator (e.g., it may be a second stimulator) that specifically binds to HVEM may be selected from the group consisting of an anti-HVEM antibody, a bivalent antibody fragment of an anti-HVEM antibody, a monovalent antibody fragment of an anti-HVEM antibody, and a proteinaceous HVEM binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment may be from any known in the art. See, e.g., WO2006054961, WO2007001459, Park et al. Cancer Immunol Immunother. 2012 Feb; 61(2):203-14.

In any of the above examples, the bivalent antibody fragment may be a (Fab)2' fragment or a bivalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment and a single chain Fv fragment (scFv). In any of the above examples, the proteinaceous binding molecule with antibody-like binding properties may be an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystal scaffold-based protein, an adnectin and an avimer.

In some aspects, the stimulatory agent specifically targets a molecule expressed on the surface of a target cell, where the molecule is a TCR, a chimeric antigen receptor, or a molecule containing an immunoreceptor tyrosine-based activation motif, or ITAM. For example, the molecule expressed on the surface of a target cell is selected from a T cell or B cell antigen receptor complex, a CD3 chain, CD3 zeta, an antigen-binding portion of a T cell receptor or a B cell receptor, or a chimeric antigen receptor. Optionally, the stimulatory agent targets a peptide:MHC class I complex.

In some embodiments, the stimulator binds to a His-tagged extracellular domain of a molecule expressed on the surface of a target cell. Optionally, the stimulator contains the peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II and shown in SEQ ID NO:8) conjugated to nickel-loaded Tris-NTA (also called His-STREPPER or His/Strep-tag® II adaptor). In some embodiments, the His-tagged molecule expressed on the surface of a target cell is CD19.

In some embodiments, the stimulatory agent specifically binds to the antibody portion of the recombinant receptor, e.g., the CAR. Optionally, the antibody portion of the recombinant receptor includes at least a portion of an immunoglobulin constant region, e.g., a hinge region, e.g., an IgG4 hinge region and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or constant portion is of a human IgG, e.g., IgG4 or IgG1. Optionally, the reagent is loaded with an αIgG that recognizes the IgG4 spacer.

In some embodiments, the desired target is a T cell receptor and/or a component of the T cell receptor. In certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a T cell costimulatory molecule, such as CD28, CD137 (4-1-BB), OX40, or ICOS.

In some embodiments, e.g., when the stimulator is not bound to a stimulating reagent (e.g., an oligomeric stimulating reagent) or a selection reagent, the stimulator is an antibody, a bivalent antibody fragment, a F(ab) ₂ , or a bivalent single chain Fv fragment. In some embodiments, when the stimulator is not bound to a reagent, the stimulator does not include binding partner C.

b. Oligomeric Stimulation Reagents As alluded to above, in certain embodiments, the stimulating reagent contains an oligomeric stimulating reagent, e.g., a streptavidin mutein reagent, conjugated, linked or attached to one or more stimulating agents. As mentioned above, in some embodiments, one or more stimulating agents are attached with a binding domain or binding partner (e.g., binding partner C) capable of binding to a specific binding site (e.g., binding site Z) of the oligomeric stimulating reagent. In some embodiments, multiple stimulating agents are reversibly bound to the oligomeric stimulating reagent. In various embodiments, the oligomeric stimulating reagent has multiple specific binding sites Z, which in certain embodiments are reversibly bound to the binding domains (e.g., binding partner C) of multiple stimulating agents. In some embodiments, the amount of bound agent is reduced or decreased in the presence of a competitor, e.g., an agent also capable of binding to the binding site (e.g., binding site Z).

In some embodiments, the oligomeric stimulatory reagent is or includes a reversible system in which at least one stimulator (e.g., a stimulator capable of generating a signal in a cell, such as a T cell) is associated, e.g., reversibly associated, with the oligomeric stimulatory reagent. Non-limiting examples of oligomeric stimulatory reagents can be found, for example, in International Published PCT Application WO 2018/197949, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the reagent contains multiple binding sites capable of binding, e.g., reversibly binding, to a stimulatory agent. Optionally, the reagent is an oligomeric stimulatory reagent to which at least one agent is attached that is capable of generating a signal (e.g., a stimulatory signal) in a cell, such as a T cell. In some embodiments, the stimulator contains at least one binding site, e.g., binding site B, that can specifically bind to an epitope or region of a molecule (e.g., a cell surface molecule or receptor), and also contains a binding partner (also referred to herein as binding partner C) that specifically binds to at least one binding site of the oligomeric stimulatory reagent, e.g., binding site Z of the reagent. In some embodiments, the binding interaction between binding partner C and at least one binding site Z is a non-covalent interaction. Optionally, the binding interaction between binding partner C and at least one binding site Z is a covalent interaction. In some embodiments, the binding interaction, such as a non-covalent interaction, between binding partner C and at least one binding site Z is reversible.

Substances that can be used as oligomer stimulating reagents in such reversible systems are known. See, e.g., U.S. Patent Nos. 5,168,049, 5,506,121, 6,103,493, 7,776,562, 7,981,632, 8,298,782, 8,735,540, 9,023,604, and published PCT applications WO2013/124474 and WO2014/076277. Non-limiting examples of reagents and binding partners capable of forming reversible interactions, as well as substances capable of reversing such binding (e.g., competitors), are described below.

In some embodiments, the oligomeric stimulating reagent is an oligomer of streptavidin, streptavidin mutein, streptavidin analog, avidin, avidin mutein, or avidin analog (e.g., neutravidin) or mixtures thereof, where such oligomeric stimulating reagent contains one or more binding sites for reversible association with the binding domain of the stimulator (e.g., binding partner C). In some embodiments, the binding domain of the stimulator can be biotin, a biotin derivative, a biotin analog, or a streptavidin-binding peptide, or other molecule capable of specifically binding to streptavidin, streptavidin mutein, streptavidin analog, avidin, avidin mutein, or avidin analog.

In certain embodiments, one or more stimulatory agents (e.g., agents capable of generating a signal in a cell, such as a T cell, e.g., those described in Section I-C-1a above) are associated with (e.g., reversibly bind to) the oligomeric stimulatory reagent, e.g., via multiple specific binding sites (e.g., binding site Z) present on the oligomeric stimulatory reagent. In some cases, this allows the stimulatory agents to be spaced closely together such that an avidity effect can occur when the agent is contacted with a target cell having (at least two copies of) a cell surface molecule that is bound or recognized by the stimulatory agent.

In some embodiments, the oligomeric stimulating reagent is a streptavidin oligomer, a streptavidin mutein oligomer, a streptavidin analog oligomer, an avidin oligomer, an avidin mutein, or an avidin analog (e.g., neutravidin), or a mixture thereof. In certain embodiments, the oligomeric stimulating reagent contains a specific binding site capable of binding to a binding domain of a stimulator (e.g., binding partner C). In some embodiments, the binding domain can be biotin, a biotin derivative, a biotin analog, or a streptavidin binding peptide, or other molecule capable of specifically binding to streptavidin, a streptavidin mutein, a streptavidin analog, avidin, an avidin mutein, or an avidin analog. Examples of streptavidin, streptavidin muteins, streptavidin analogs, avidin, avidin muteins or avidin analogs (e.g., neutravidin) contemplated for inclusion in the oligomeric stimulation reagent system, and avidin-binding domain molecules such as biotin, biotin derivatives, biotin analogs, or streptavidin-binding peptides, or other molecules capable of specifically binding to streptavidin, streptavidin muteins, streptavidin analogs, avidin, avidin muteins or avidin analogs, are described in Section II-A below. Additionally, in the methods provided herein, it is contemplated that the oligomeric stimulating reagent may include a molecule capable of binding to an oligohistidine affinity tag, glutathione-S-transferase, calmodulin or an analog thereof, calmodulin-binding peptide (CBP), FLAG-peptide, HA tag, maltose-binding protein (MBP), an HSV epitope, a myc epitope, and/or a biotinylated carrier protein (see Section II-A).

In particular embodiments, provided herein are oligomeric stimulating reagents that are composed of and/or contain a plurality of streptavidin tetramers or streptavidin mutein tetramers. In certain embodiments, the oligomeric stimulating reagents provided herein contain a plurality of binding sites that reversibly bind or have the ability to reversibly bind one or more stimuli. In some embodiments, the oligomeric stimulating reagents have a radius, e.g., average radius, of 70 nm to 125 nm, inclusive, a molecular weight of 1×10 ⁷ g/mol to 1×10 ⁹ g/mol, inclusive, and/or 1,000 to 5,000 streptavidin tetramers or streptavidin mutein tetramers, inclusive. In some embodiments, the oligomeric stimulating reagents are bound (e.g., reversibly bound) to one or more stimuli, e.g., agents that bind to molecules (e.g., receptors) on the surface of a cell. In certain embodiments, the one or more stimulatory agents are agents described herein, for example, in section IC-1a. In some embodiments, the one or more stimulatory agents contain a monovalent binding site (e.g., binding site B). In some embodiments, the monovalent binding site binds CD3. In some embodiments, the monovalent binding site binds a costimulatory molecule, such as those described herein. In some embodiments, the monovalent binding site binds CD28. In some embodiments, the one or more stimulatory agents contain a monovalent binding site capable of binding to CD3 and/or CD28. In some embodiments, the stimulatory agent is an anti-CD3 antibody and/or an anti-CD28 antibody or an antigen-binding fragment thereof, such as a binding partner C, for example, an antibody or an antigen-binding fragment thereof that contains a streptavidin-binding peptide, for example, Strep-tag® II. In certain embodiments, the one or more stimulatory agents are binding partners, for example, anti-CD3 Fab and/or anti-CD28 Fab that contain a streptavidin-binding peptide, for example, Strep-tag® II. In certain embodiments, the one or more agents comprise streptavidin-based oligomers, such as streptavidin mutein oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab. In some embodiments, the oligomeric stimulating reagent is any of those described in WO2015/158868 or WO2018/197949.

In some embodiments, provided herein are oligomeric stimulating reagents that are composed of and/or contain a plurality of streptavidin tetramers or streptavidin mutein tetramers. In certain embodiments, the oligomeric stimulating reagents provided herein contain a plurality of binding sites that reversibly bind or have the ability to reversibly bind one or more stimuli. In some embodiments, the oligomeric particles have a radius, e.g., average radius, of 80 nm to 120 nm, inclusive, a molecular weight, e.g., average molecular weight, of 7.5×10 ⁶ g/mol to 2×10 ⁸ g/mol, inclusive, and/or an amount, e.g., average amount, of 500 to 10,000, inclusive, of streptavidin tetramers or streptavidin mutein tetramers. In some embodiments, the oligomeric stimulatory reagent is conjugated (e.g., reversibly conjugated) to one or more stimuli, e.g., agents that bind to molecules (e.g., receptors) on the surface of cells. In certain embodiments, the one or more stimuli are agents described herein, e.g., in section IB-2-a. In some embodiments, the stimuli are anti-CD3 and/or anti-CD28 antibodies or antigen-binding fragments thereof, e.g., binding partner C, e.g., an antibody or antigen-binding fragment thereof that contains a streptavidin-binding peptide, e.g., Strep-tag® II. In particular embodiments, the one or more stimuli are binding partners, e.g., anti-CD3 Fab and/or anti-CD28 Fab that contain a streptavidin-binding peptide, e.g., Twin-Strep-tag (e.g., SEQ ID NO:16).

In some embodiments, cells are ^administered at 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 1.2 μg, 1.4 μg, 1.6 μg, 1.8 μg, 2 μg, 2.2 μg, 2.4 μg, 2.6 μg, 2.8 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg or 10 μg, or about 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 1.2 μg, 1.4 μg, 1.6 μg, 1 0.8μg, 2μg, 2.2μg, 2.4μg, 2.6μg, 2.8μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg or 10μg, or at least 0.01μg, 0.02μg, 0.03μg, 0.04μg, 0.05μg, 0.1μg, 0.2μg, 0.3μg, 0.4μg, 0.5μg, 0.75μg, 1μg, 1.2μg, 1.4μg, 1.6μg, 1.8μg, 2μg, 2.2μg, 2.4μg, 2.6μg, 2.8μg, 3μg, 4μg, 5μg, 6μg, 7μg, 8μg, 9μg or 10μg of oligomeric stimulating reagent (e.g., Strep-tagged anti-CD3 In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 4 μg or about 4 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 ⁶ cells. In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 3 μg or about 3 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per ^{10 6} cells. In some embodiments, ^the cells are stimulated or subjected to stimulation in the presence of 2.75 μg or about 2.75 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 ⁶ cells. In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 2.5 μg or about 2.5 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 6 cells. In some embodiments, ^the cells are stimulated or subjected to stimulation in the presence of 2.25 μg or about 2.25 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 ⁶ cells. In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 2 μg or about 2 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 6 cells. In particular embodiments, ^the cells are stimulated or subjected to stimulation in the presence of 1.8 μg or about 1.8 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 ⁶ cells. In particular embodiments, the cells are stimulated or subjected to stimulation in the presence of 1.6 μg or about 1.6 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 6 cells. In a particular embodiment, ^the cells are stimulated or subjected to stimulation in the presence of 1.4 μg or about 1.4 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 ⁶ cells. In a particular embodiment, the cells are stimulated or subjected to stimulation in the presence of 1.2 μg or about 1.2 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 6 cells. In particular embodiments, ^the cells are stimulated or subjected to stimulation in the presence of 1 μg or about 1 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 ⁶ cells. In particular embodiments, the cells are stimulated or subjected to stimulation in the presence of 0.8 μg or about 0.8 μg of oligomeric stimulation reagent (e.g., streptavidin-based oligomers conjugated to Strep-tagged anti-CD3 Fab and Strep-tagged anti-CD28 Fab, e.g., streptavidin mutein oligomers) per 10 6 cells. ^In some embodiments, cells are stimulated or subjected to ^stimulation in the ^presence of ^10x108 , 9x108, ^8x108 , ^{7x108 , 6x108} , 5x108, 4x108 ^, 3x108 ^, 2x108, ^1x108 , or about 10x108, 9x108 ^, 8x108 ^, 7x108, ^6x108 , ^5x108 , ^4x108 , ^3x108 , ^2x108 , ^1x108 of oligomeric stimulation reagent ^. In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 7× ¹⁰ , 6× ¹⁰ , 5× ¹⁰ , 4× ¹⁰ , 3×10 ^, or about 7× ¹⁰ , 6× ¹⁰ , 5×10 ^, 4× ¹⁰ , 3×10 ^. In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 7× ¹⁰ to 3× ¹⁰ , or about 7× ¹⁰ to 3× ¹⁰ . In some embodiments, the cells are stimulated or subjected to stimulation in the presence of 6× ¹⁰ to 4× ¹⁰ , or about 6× ¹⁰ to 4× ¹⁰ . In some embodiments, the cells are stimulated or subjected to stimulation in the presence of, or about ^, 6× ^{10 8 to} 5×10 ⁸ of the oligomeric stimulating reagent. In some embodiments, the cells are stimulated or subjected to stimulation in the presence of, or about ^{, 5×10 8 of} the oligomeric stimulating reagent.

In some embodiments, cells, e.g., selected cells of a sample, are stimulated or subjected to stimulation at a ratio of oligomeric stimulating reagent to cells of 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1 or about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In certain embodiments, the ratio of oligomeric stimulating reagent to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, between 1.1:1 and 0.9:1. In certain embodiments, the ratio of oligomeric stimulating reagent to cells is about 1:1 or is 1:1. In certain embodiments, the ratio of oligomeric stimulating reagent to cells is about 0.3:1 or is 0.3:1. In certain embodiments, the ratio of oligomeric stimulating reagent to cells is about 0.2:1 or is 0.2:1.

In certain aspects, the mass ratio between the oligomer particles and the attached agent in the oligomeric stimulation reagent is about 3:1. In certain aspects, the mass ratio between the oligomer particles and the attached anti-CD3 Fab and the attached anti-CD28 Fab in the oligomeric stimulation reagent is about 3:0.5:0.5. In certain aspects, 4 μg of the oligomeric stimulation reagent is or comprises 3 μg of oligomer particles and 1 μg of attached agent, such as 0.5 μg of anti-CD3 Fab and 0.5 μg of anti-CD28 Fab. In another example, 1.2 μg of the oligomeric stimulation reagent per 10 ⁶ cells is or comprises 0.9 μg of oligomer particles and 0.3 μg of attached agent, such as 0.15 μg of anti-CD3 Fab and 0.15 μg of anti-CD28 Fab, per 10 ⁶ cells. In some embodiments, the oligomeric stimulation reagent is added to serum-free medium and stimulation is performed in serum-free medium, e.g., as described in Section III of the present specification or in PCT/US2018/064627.

In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T Cell Expansion Basal Medium (ThermoFisher). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion and/or activation of cells (e.g., T cells), such as those provided by additional supplements (e.g., OpTmizer™ T Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium comprises a serum replacement supplement, such as immune cell serum replacement, e.g., ThermoFisher, #A2596101, CTS™ immune cell serum replacement, or Smith et al. Clin Transl Immunology. 2015 Jan;4(1):e31. In some embodiments, the serum-free medium further comprises a free amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15.

D. Elution In aspects of the methods provided herein, the elution of cells, such as target cells (e.g., T cells), from a chromatography column following incubation with a stimulating agent is accomplished without using a competitor or free binder, as described herein.In some embodiments, during incubation with a stimulating agent, the cells immobilized on a chromatography matrix (e.g., stationary phase) via a selection agent spontaneously detach from the selection agent.In some embodiments, spontaneous detachment occurs within 1 day from the start of incubation with a stimulating agent.In some embodiments, spontaneous detachment occurs within 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 hours from the start of incubation with a stimulating agent. In some embodiments, spontaneous detachment occurs within about 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 19-24, 20-24, 21-24, 22-24, 23-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 hours after initiation of incubation with the stimuli. In some embodiments, detachment from the column occurs within 4-5 hours or within about 4-5 hours, e.g., within 4.5 hours, after the start of incubation with the stimulating agent. In some embodiments, a majority of the plurality of target cells (e.g., T cells) immobilized on a chromatography matrix (e.g., stationary phase) via a selection substance detaches in less than 1 day from the start of incubation with the stimulating agent. In some embodiments, a majority of the plurality of target cells (e.g., T cells) immobilized on a chromatography matrix (e.g., stationary phase) via a selection substance detaches in less than 24 hours from the start of incubation with the stimulating agent. In some embodiments, a majority of the plurality of target cells (e.g., T cells) immobilized on a chromatography matrix (e.g., stationary phase) via a selection substance detaches in less than 12 hours from the start of incubation with the stimulating agent. In some embodiments, a majority of the plurality of target cells (e.g., T cells) immobilized on a chromatography matrix (e.g., stationary phase) via a selection substance detaches in less than 5 hours from the start of incubation with the stimulating agent. In some embodiments, a majority of the target cells (e.g., T cells) immobilized on a chromatography matrix (e.g., stationary phase) via a selection agent detach in less than 4 hours from the start of incubation with a stimulant. In some embodiments, a majority of the target cells (e.g., T cells) immobilized on a chromatography matrix (e.g., stationary phase) via a selection agent detach in less than 2 hours from the start of incubation with a stimulant.

In some embodiments, the spontaneously detached cells are eluted and/or collected from the chromatography column by gravity. In some embodiments, the spontaneously detached cells are eluted from the chromatography column using a wash step. In some embodiments, at least one wash step is performed after, or about, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours from the start of incubation with the stimulant or a stimulating reagent containing the stimulant. In some embodiments, one or more wash steps are performed after, or about, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours from the start of incubation with the stimulant or a stimulating reagent containing the stimulant. In some embodiments, one or more washing steps are performed after about 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 7 to 24, 8 to 24, 9 to 24, 10 to 24, 11 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 20 to 24, 22 to 24, 24 to 24, 26 to 24, 28 to 24, 29 to 30, 31 to 31, 32 to 32, 33 to 33, 34 to 34, 35 to 35, 36 to 36, 37 to 37, 38 to 38, 39 to 40, 41 to 41, 42 to 42, 43 to 43, 44 to 44, 45 to 45, 46 to 46, 47 to 47, 48 to 48, 49 to 50, 51 to 51, 52 to 53, 54 to 55, 56 to 57, 58 to 59, 59 to 60, 61 to 62, 62 to 63, 63 to 64, 64 to 65, 65 to 66, 66 to 67, 67 to 68, 68 to 69, 70 to 71, 72 to 73, 74 to 75, 75 to 76, 76 to 77, 77 to 78, 78 to 79, 79 to 80, 8 It will be carried out within 18-24, 19-24, 20-24, 21-24, 22-24, 23-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 or 2-3 hours.

In some embodiments, the elution and/or collection step after the selection and on-column stimulation step is performed within 2 days or about 2 days after applying the sample to a chromatography column (e.g., stationary phase), e.g., as described in Section I-A. In some embodiments, the elution and/or collection step after the selection and on-column stimulation step is performed within 1-2 days or about 1-2 days after applying the sample to a chromatography column (e.g., stationary phase), e.g., as described in Section I-A. In some embodiments, the elution and/or collection step after the selection and on-column stimulation step is performed within 1 day or about 1 day after applying the sample to a chromatography column (e.g., stationary phase), e.g., as described in Section I-A. In some embodiments, the elution and/or collection step after the selection and on-column stimulation step is performed less than 1 day after applying the sample to a chromatography column (e.g., stationary phase), e.g., as described in Section I-A. In some embodiments, the elution and/or collection step after the selection and on-column stimulation steps is performed within or about 48, 36, 24, 12, 6, 4, or 2 hours, inclusive, after the sample is applied to the chromatographic column (e.g., stationary phase), e.g., as described in Section I-A. In some embodiments, the collection or elution step after the selection and on-column stimulation steps is performed within 2-48, 2-36, 2-24, 2-12, 2-6, 2-4, 4-48, 4-36, 4-24, 4-12, 4-6, 6-48, 6-36, 6-24, 6-12, 12-48, 12-36, 1 The process is carried out within 2-24, 24-48, 24-36, or 36-48 hours or within about 2-48, 2-36, 2-24, 2-12, 2-6, 2-4, 4-48, 4-36, 4-24, 4-12, 4-6, 6-48, 6-36, 6-24, 6-12, 12-48, 12-36, 12-24, 24-48, 24-36, or 36-48 hours. In some embodiments, the process duration, including steps from selection and on-column stimulation to elution or collection, is less than 48, 36, 24, 12, 6, 4, or 2 hours. In some embodiments, the process duration, including steps from selection and on-column stimulation to elution or collection, is less than 36 hours. In some embodiments, the process duration, including steps from selection and on-column stimulation to elution or collection, is less than 24 hours. In some embodiments, the process duration including the steps of selection and on-column stimulation to elution or collection is less than 12 hours. In some embodiments, the process duration including the steps of selection and on-column stimulation to elution or collection is 7 hours, about 7 hours, or less than 7 hours. In some embodiments, the process duration including the steps of selection and on-column stimulation to elution or collection is 6.5 hours, about 6.5 hours, or less than 6.5 hours. In some embodiments, the process duration including the steps of selection and on-column stimulation to elution or collection is 6 hours, about 6 hours, or less than 6 hours. In some embodiments, the process duration including the steps of selection and on-column stimulation to elution or collection is 5.5 hours, about 5 hours, or less than 5 hours. In some embodiments, the process duration including the steps of selection and on-column stimulation to elution or collection is 4.5 hours, about 4.5 hours, or less than 4.5 hours. In some embodiments, the process duration, including steps from selection and on-column stimulation to elution or collection, is 4 hours, about 4 hours, or less than 4 hours.

In some embodiments, the wash medium is a culture medium. Thus, in some embodiments, the eluted cells can proceed directly to downstream processing (e.g., subsequent selection steps, stimulation steps, incubation steps, genetic manipulation). In some embodiments, the wash medium comprises a serum-free basal medium containing glutamine and recombinant IL-2, IL-15, and IL-7. In some embodiments, the wash medium comprises a serum-free basal medium containing glutamine and lacking one or more of recombinant IL-2, IL-15, and IL-7. In some embodiments, the wash medium comprises a serum-free basal medium lacking glutamine and one or more of recombinant IL-2, IL-15, and IL-7.

In some embodiments, the eluate includes a stimulating reagent (e.g., an oligomeric stimulating reagent). In some embodiments, the collected cells are still bound to the stimulating agent (e.g., a stimulating agent bound to the oligomeric stimulating reagent). In some embodiments, the stimulating agent contained in the eluate is bound to the eluted cells and the stimulating reagent (e.g., an oligomeric stimulating reagent). Thus, the collected and/or eluted cells may still be considered to be under stimulating conditions. In some embodiments, the detached and eluted cells are under stimulating conditions (e.g., still stimulated). In some embodiments, the eluted cells may continue to be under stimulating conditions, e.g., as described in Sections I-C.

In some embodiments, the column and collection vessel are connected in a closed system. In some embodiments, the closed system is sterile. In some embodiments, the selection, stimulation and elution steps are performed by an automated system with little or no manual, e.g., human, manipulation or intervention.

E. Genetic Engineering In some embodiments, the methods provided include genetically engineering the cells (e.g., the output composition of the selected stimulated cells), e.g., by introducing a heterologous or recombinant polynucleotide encoding a recombinant protein. Such recombinant proteins can include recombinant receptors, e.g., those described in Section IV. Introduction of a polynucleotide encoding a recombinant protein, e.g., a heterologous or recombinant polynucleotide, into the cells can be carried out using any of a number of known vectors. Such vectors include viral systems, e.g., lentiviral and gamma retroviral systems. Exemplary methods include methods for transferring a heterologous polynucleotide encoding a receptor, including by viral transduction, e.g., retroviral or lentiviral transduction. In some embodiments, the population of stimulated cells (e.g., the output composition of the selected stimulated cells) is genetically engineered, e.g., by introducing a heterologous or recombinant polynucleotide encoding a recombinant receptor, thereby producing a population of transformed cells (also referred to herein as a population of transformed cells).

In certain embodiments, the cells (e.g., T cells, CD3+, CD4+, CD8+ T cells) are genetically engineered, transformed, or transduced after the cells have been subjected to on-column stimulation, such as by any of the methods provided herein, e.g., in Sections I-C. In certain embodiments, one or more stimulated populations have been previously cryoprotected and stored, and are thawed before the cells are genetically engineered, transformed, transfected, or transduced.

In certain embodiments, cells (e.g., T cells, CD3+, CD4+, CD8+ T cells) are genetically engineered, transformed or transduced after the cells have been stimulated or subjected to stimulation or cultured under stimulatory conditions (e.g., on-column stimulation). In certain embodiments, the cells are genetically engineered, transformed or transduced at or within about 72, 60, 48, 36, 24, 12, 5, 4 or 2 hours from the start of stimulation. In some embodiments, the cells are genetically engineered at or within about 2, 3, 4, 5 or 6 hours from the start of on-column stimulation. In some embodiments, the cells are engineered at or about 4-5 hours from the start of on-column stimulation. In some embodiments, the cells are still under stimulation conditions during the genetic engineering. In certain embodiments, the cells are engineered, transformed or transduced between or about 2-6 hours or 6-12 hours after the start of stimulation. In certain embodiments, the cells are engineered, transformed or transduced between or about 12-48 hours, 16-36 hours or 18-30 hours after the start of stimulation. In particular embodiments, the cells are engineered, transformed or transduced between or about 18-30 hours after the start of stimulation. In particular embodiments, the cells are engineered, transformed or transduced between or about 22-24 hours after the start of stimulation. In certain embodiments, the cells are engineered, transformed or transduced at or about 6 or 12 hours after the start of stimulation. In certain embodiments, the cells are engineered, transformed or transduced at or about 4 or 5 hours after the start of stimulation. In certain embodiments, the cells are engineered, transformed or transduced at or about 2 or 3 hours after the start of stimulation.

In certain embodiments, the method for genetic engineering is performed by contacting or introducing into one or more cells of the population (e.g., the output composition of the selected and stimulated cells) a nucleic acid molecule or polynucleotide encoding a recombinant protein, e.g., a recombinant receptor. In certain embodiments, the nucleic acid molecule or polynucleotide is heterologous to the cell. In certain embodiments, the heterologous nucleic acid molecule or heterologous polynucleotide is not native to the cell. In certain embodiments, the heterologous nucleic acid molecule or heterologous polynucleotide encodes a protein, e.g., a recombinant protein, that is not naturally expressed by the cell. In certain embodiments, the heterologous nucleic acid molecule or polynucleotide is or contains a nucleic acid sequence that is not found in the cell prior to the contact or introduction.

In some embodiments, the cells, e.g., the output composition, are engineered, e.g., transduced, in the presence of a transduction adjuvant. Exemplary transduction adjuvants include, but are not limited to, polycations, fibronectin or fibronectin-derived fragments or variants, and RetroNectin. In certain embodiments, the cells are engineered in the presence of a polycation, fibronectin or fibronectin-derived fragments or variants, and/or RetroNectin. In certain embodiments, the cells are engineered in the presence of a polycation that is polybrene, DEAE-dextran, protamine sulfate, poly-L-lysine, or cationic liposomes. In certain embodiments, the cells are engineered in the presence of protamine sulfate.

In some embodiments, the genetic manipulation, e.g., transduction, is performed in a serum-free medium, e.g., as described in Section III herein or PCT/US2018/064627. In some embodiments, the serum-free medium is a defined or precisely defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, e.g., filtered to remove inhibitors and/or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins and/or attachment factors. In some embodiments, the medium contains glutamine.

In certain embodiments, the cells are engineered in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In certain embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind and/or are capable of binding to a receptor expressed by and/or endogenous to the T cell. In certain embodiments, the one or more cytokines are or include members of the four-alpha-helical bundle family of cytokines. In some embodiments, members of the four-alpha-helical bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include recombinant IL-2.

In certain embodiments, the cells, e.g., stimulated cells, are engineered, e.g., transduced, under stimulatory conditions in the presence of IL-2, IL-7, and/or IL-15. In certain embodiments, the IL-2, IL-7, and/or IL-15 are recombinant. In certain embodiments, the IL-2, IL-7, and/or IL-15 are human. In certain embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and/or IL-15. In certain embodiments, the cells are engineered, e.g., transduced, under stimulatory conditions in the presence of recombinant IL-2, IL-7, and IL-15.

In some embodiments, the cells are engineered, transformed, or transduced in the presence of the same or similar medium that was present during stimulation. In some embodiments, the cells are engineered, transformed, or transduced in medium that has the same cytokines as the medium that was present during stimulation. In certain embodiments, the cells are engineered, transformed, or transduced in medium that has the same cytokines at the same concentrations as the medium that was present during stimulation.

1. Transduction In some embodiments, genetically engineering a cell (e.g., an output composition) is or includes introducing a polynucleotide, e.g., a heterologous or recombinant polynucleotide, into a cell by transduction. In some embodiments, the cell is transduced or subjected to transduction using a viral vector. In certain embodiments, the cell is transduced or subjected to transduction using a viral vector. In some embodiments, the virus is a retroviral vector, such as a gamma retroviral vector or a lentiviral vector. Lentiviral transduction methods are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

In some embodiments, transduction is performed by contacting one or more cells of the population (e.g., the output composition) with a nucleic acid molecule encoding a recombinant protein, e.g., a recombinant receptor. In some embodiments, the contacting can be accomplished using centrifugation, e.g., spinoculation (e.g., centrifuge inoculation). Such methods include any of the methods as described in International Publication No. WO2016/073602. Exemplary centrifuge chambers include those manufactured and sold by Biosafe SA, including centrifuge chambers for use with the Sepax® and Sepax® 2 systems, including the A-200/F and A-200 centrifuge chambers, as well as various kits for use with such systems. Exemplary chambers, systems, and processing devices and cabinets are described, for example, in U.S. Pat. No. 6,123,655, U.S. Pat. No. 6,733,433, and published U.S. patent application publication number US2008/0171951, and published International patent application publication number WO00/38762, the contents of each of which are incorporated herein by reference in their entirety. Exemplary kits for use with such systems include, but are not limited to, disposable kits sold by BioSafe SA under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

In some embodiments, the provided method is used in conjunction with transducing 300×10 ⁶ , about 300×10 6 , or less than 300×10 ⁶ cells, e.g., viable T cells, of the stimulated cell population with a viral vector containing a polynucleotide encoding a recombinant receptor. In certain embodiments, 100×10 ⁶ or about 100×10 ⁶ cells, e.g., viable T cells, of the stimulated cell population are transduced or subjected to transduction. In some embodiments, 1×10 ⁶ cells per mL, e.g., viable T cells of the stimulated cell population, are transduced or subjected to transduction. In some embodiments, the dose of viral vector is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μL or about 10, 9, 8, ⁷ , 6, 5, 4, 3, 2, or 1 μL per 1×10 ⁶ cells. In some embodiments, the dose of the viral vector is at or about 6-4 μL per 1× ¹⁰ cells. In some embodiments, the dose of the viral vector is at or about 5 μL per 1× ¹⁰ cells.

In some embodiments, transduction is performed in serum-free medium. In some embodiments, transduction is performed in the presence of IL-2, IL-7 and IL-15. In some embodiments, the viral vector for transduction is frozen and thawed prior to use, and the thawed viral vector is diluted in serum-free medium. In some embodiments, the dilution of the viral vector and the serum-free medium for transduction are as described in Section III of this specification or PCT/US2018/064627.

In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements (e.g., OpTmizer™ T Cell Expansion Basal Medium (ThermoFisher). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more additional components for the maintenance, expansion and/or activation of cells (e.g., T cells), such as those provided by additional supplements (e.g., OpTmizer™ T Cell Expansion Supplement (ThermoFisher)). In some embodiments, the serum-free medium comprises a serum replacement supplement, such as immune cell serum replacement, e.g., ThermoFisher, #A2596101, CTS™ immune cell serum replacement, or Smith et al. Clin Transl Immunology. 2015 Jan;4(1):e31. In some embodiments, the serum-free medium further comprises a free amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium further comprises one or more recombinant cytokines, such as recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15.

In certain embodiments, the cells, e.g., cells of a stimulated cell population (e.g., output composition), contain at least 80%, at least 85%, at least 90%, or at least 95% cells that are CD4+ T cells or CD8+ T cells. In some embodiments, transduction is performed for 24-48 hours, 36-12 hours, 18-30 hours, or about 24 hours, including post-transduction incubation. In some embodiments, transduction is performed for 72 hours ± 6 hours or about 72 hours ± 6 hours, including post-transduction incubation. In some embodiments, transduction is performed for 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours. In some embodiments, transduction is performed for 0.5, 1, 1.5, or 2 hours or for about 0.5, 1, 1.5, or 2 hours. In some embodiments, transduction is performed for 0.5 to 1.5 hours or for about 0.5 to 1.5 hours. In some embodiments, transduction is performed for 1 hour or for about 1 hour.

In certain embodiments, the transduction step is initiated within 2 days, 36 hours, 30 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, or 2 hours from the onset or start of incubation, e.g., incubation under stimulatory conditions. In certain embodiments, the transduction step is initiated within 4-5 hours from the onset or start of incubation, e.g., incubation under stimulatory conditions. In certain embodiments, the transduction step is initiated at about 20 hours from the onset or start of incubation, e.g., incubation under stimulatory conditions. In certain embodiments, the transduction step is initiated at 4-5 hours or about 4-5 hours from the onset or start of incubation, e.g., incubation under stimulatory conditions.

In some embodiments, the system includes and/or is disposed in association with other equipment, including equipment for operating, automating, controlling, and/or monitoring aspects of the transduction process and one or more various other process steps performed in the system, such as one or more process steps that may be performed with or in association with a centrifuge chamber system as described herein or in International Publication No. WO2016/073602. In some embodiments, the equipment is contained within a cabinet. In some embodiments, the equipment includes a cabinet that includes a housing containing control circuitry, a centrifuge, a cover, a motor, a pump, sensors, a display, and a user interface. Exemplary devices are described in U.S. Pat. No. 6,123,655, U.S. Pat. No. 6,733,433, and U.S. Pat. No. 2008/0171951.

In some embodiments, the system includes a series of containers, e.g., bags, tubes, stopcocks, clamps, connectors, and centrifugation chambers. In some embodiments, the containers, e.g., bags, include one or more containers, e.g., bags, that contain the cells to be transduced and the viral vector particles, either in the same container or in separate containers, e.g., in the same bag or in separate bags. In some embodiments, the system further includes one or more containers, e.g., bags, that contain media, e.g., diluents and/or wash solutions, that are drawn into the chambers and/or other components to dilute, resuspend, and/or wash the components and/or populations during the method. The containers can be connected to one or more locations in the system, e.g., locations that correspond to input lines, diluent lines, wash lines, waste lines, and/or output lines.

In some embodiments, the chamber is associated with a centrifuge that is capable of achieving rotation of the chamber, such as around its axis of rotation. Rotation can occur before, during, and/or after incubations associated with transduction of cells, and/or in one or more of the other processing steps. Thus, in some embodiments, one or more of the various processing steps are performed under rotation, e.g., at a particular force. The chamber is typically rotatable in a vertical or near vertical direction, such that the chamber is positioned vertically during centrifugation, with the side walls and axis being vertical or near vertical, and the end walls being horizontal or near horizontal.

In some embodiments, the cell-containing population and the viral vector particle-containing population and optionally air can be combined or mixed before providing the populations to the cavity. In some embodiments, the cell-containing population and the viral vector particle-containing population and optionally air are provided separately and combined and mixed in the cavity. In some embodiments, the cell-containing population, the viral vector particle-containing population and optionally air can be provided to the internal cavity in any order. In any of such some embodiments, the cell- and viral vector particle-containing population is an input population that is combined or mixed together once, regardless of whether it is combined or mixed inside or outside the centrifuge chamber and/or whether the cells and viral vector particles are provided to the centrifuge chamber together or separately, e.g., simultaneously or sequentially.

In some embodiments, the introduction of a volume of gas, such as air, occurs prior to incubation of the cells and viral vector particles, such as rotation in a transduction method. In some embodiments, the introduction of a volume of gas, such as air, occurs during incubation of the cells and viral vector particles, such as rotation in a transduction method.

In some embodiments, the liquid volume of cells or viral vector particles, and optionally the volume of air, that make up the transduced population can be a predetermined volume. The volume can be a volume programmed into and/or controlled by circuitry associated with the system.

In some embodiments, the uptake of the transduced population, and optionally the gas, such as air, is controlled manually, semi-automatically, and/or automatically until a desired or predetermined volume is uptaken into the internal cavity of the chamber. In some embodiments, a sensor associated with the system can detect the liquid and/or gas entering or leaving the centrifuge chamber via its color, flow rate, and/or density, etc., and communicate with associated circuitry to stop or continue the uptake as needed until such a desired or predetermined volume of uptake is achieved. In some aspects, a sensor can be made that is programmed or capable of detecting only liquid in the system and not gas (e.g., air), allowing a gas, such as air, to pass through the system without stopping the uptake. In some such embodiments, a piece of non-transparent tubing can be placed in the line near the sensor while uptake of the gas, such as air, is desired. In some embodiments, the uptake of the gas, such as air, can be manually controlled.

In aspects of the methods provided, the interior cavity of the centrifuge chamber is subjected to high speed rotation. In some embodiments, the rotation is accomplished prior to, simultaneously with, subsequent to, or intermittently with the incorporation of the liquid input mass and, optionally, air. In some embodiments, the rotation is accomplished subsequent to the incorporation of the liquid input mass and, optionally, air. In some embodiments, the rotation is at or about 200g, 300g, 400g, 500g, 600g, 700g, 800g, 1000g, 1100g, 1500, 1600g, 1800g, 2000g, 2200g, 2500g, 3000g, 3200g, 3500g, or 4000g on the inner surface of the sidewall of the internal cavity and/or on the surface layer of the cells. g, or at least about or at least 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 1000 g, 1100 g, 1500, 1600 g, 1800 g, 2000 g, 2200 g, 2500 g, 3000 g, 3200 g, 3500 g, or 4000 g. In some embodiments, the rotation is by centrifugation at a force that is greater than or about 1100 g, such as greater than or about 1200 g, greater than or about 1400 g, greater than or about 1600 g, greater than or about 1800 g, greater than or about 2000 g, greater than or about 2400 g, greater than or about 2800 g, greater than or about 3000 g, or greater than or about 3200 g. In certain embodiments, the centrifugation rotation is a rotation at a force between 600 g and 800 g. In certain embodiments, the centrifugation rotation is a rotation at a force of 693 g or about 693 g. In some embodiments, the centrifugation rotation is a rotation at a force of 1600 g or about 1600 g.

In some embodiments, the gas, such as air, in the cavity of the chamber is vented from the chamber. In some embodiments, the gas, such as air, is vented to a container operably connected to the centrifuge chamber as part of a closed system. In some embodiments, the container is an open or empty container. In some embodiments, the air, such as the gas in the cavity of the chamber, is vented through a filter operably connected to the interior cavity of the chamber via a sterile line. In some embodiments, the air is vented using a manual, semi-automated, or automated process. In some embodiments, the air is vented from the chamber prior to, simultaneously with, intermittently with, or subsequent to squeezing the output population containing incubated cells and viral vector particles, e.g., transduction-initiated cells or cells transduced by a viral vector, from the cavity of the chamber.

In some embodiments, transduction and/or other incubations are performed as or as part of a continuous or semi-continuous process. In some embodiments, a continuous process involves continuous uptake of cells and viral vector particles, e.g., transduction composition (either as a single pre-existing composition or by continuously drawing into the same container, e.g., cavity, thereby mixing the portions), and/or continuous squeezing or expulsion of liquid, and optionally gas (e.g., air) from the container, during at least a portion of the incubation, e.g., centrifugation. In some embodiments, the continuous uptake and continuous squeezing are performed at least partially simultaneously. In some embodiments, the continuous uptake is performed during a portion of the incubation, e.g., during a portion of the centrifugation, and the continuous squeezing is performed during another portion of the incubation. The two may alternate. Thus, by continuous uptake and squeezing, a larger overall volume of sample can be processed, e.g., transduced, while the incubation is performed.

In some embodiments, the incubation is part of a continuous process, and the method includes achieving continuous uptake of the transduction composition into the cavity during at least a portion of the incubation, achieving continuous squeezing of the liquid during rotation of the chamber and during a portion of the incubation, and optionally evacuating gas (e.g., air) from the cavity through at least one opening during rotation of the chamber.

In some embodiments, the semi-continuous incubation is performed by alternating between taking a composition into the cavity, incubating, squeezing liquid from the cavity, and optionally expelling gas (e.g., air) from the cavity into an output container or the like, then taking a subsequent (e.g., second, third, etc.) composition containing more cells and other reagents for processing, such as viral vector particles, and repeating the process. For example, in some embodiments, the incubation is part of a semi-continuous process, and the method includes, prior to incubation, achieving taking a transduction composition into the cavity through at least one opening, and following incubation, achieving squeezing fluid from the cavity; achieving taking another transduction composition into the internal cavity, including cells and viral vector particles; and incubating another transduction composition in the internal cavity under conditions in which the cells in the other transduction composition are transduced or subjected to transduction by the vector. This process can be continued iteratively for several additional rounds. In this regard, semi-continuous or continuous methods may allow for the production of even larger volumes and/or numbers of cells.

In some embodiments, a portion of the transduction incubation is performed in a centrifuge chamber and under conditions that include rotation or centrifugation.

In certain embodiments, transduction of cells with a viral vector is or includes spinoculation, e.g., centrifugation, of a mixture containing cells and viral particles. In some embodiments, the composition containing cells and viral particles can be spun at a relatively low force or speed, such as at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm, or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), generally slower than the speed used to pellet the cells. In some embodiments, the rotation is, for example, between 100g and 4000g, or about 100g and 4000g (e.g., between 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, 1500g, 2000g, 2500g, 3000g, or 3500g, or about 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, 1500g, 2000g, or 3500g). , 2500g, 3000g or 3500g, or at least about 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, 1500g, 2000g, 2500g, 3000g or 3500g), e.g., relative centrifugal force.

In some embodiments, the cells are spun with the viral vector at a force, e.g., a relative centrifugal force, of 100 g to 4000 g, 200 g to 1,000 g, 500 g to 1200 g, 1000 g to 2000 g, 600 g to 800 g, 1200 g to 1800 g, or 1500 g to 1800 g or about 100 g to 4000 g, 200 g to 1,000 g, 500 g to 1200 g, 1000 g to 2000 g, 600 g to 800 g, 1200 g to 1800 g, or 1500 g to 1800 g. In certain embodiments, the cells are cultured at least 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, 1200g, 1500g, 1600g, 2000g, 2500g, 3000g, 3200g, or 3500g, inclusive, together with the viral vector particles. The cells are spinoculated at or about 0g, 1000g, 1200g, 1500g, 1600g, 2000g, 2500g, 3000g, 3200g, or 3500g. In some embodiments, the cells are transduced or subjected to transduction with the viral vector at a force of at or about 692g. In certain embodiments, the cells are transduced or subjected to transduction with the viral vector at a force of at or about 1600g. In some embodiments, the force is at the inner surface of the sidewall of the internal cavity and/or at a surface layer of the cells.

In certain embodiments, the cells are spinoculated, e.g., the cell composition containing the cells and the viral vector is rotated for greater than or about 5 minutes, e.g., greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes, or greater than or about 120 minutes; or for 5 to 120 minutes, 30 to 90 minutes, 15 to 60 minutes, 15 to 45 minutes, 30 to 60 minutes, or 45 to 60 minutes, or for about 5 to 120 minutes, 30 to 90 minutes, 15 to 60 minutes, 15 to 45 minutes, 30 to 60 minutes, or 45 to 60 minutes, inclusive. In some embodiments, the cells are spinoculated with the viral vector for 30 minutes or about 30 minutes. In certain embodiments, the cells are spinoculated with the viral vector for 60 minutes or about 60 minutes.

In some embodiments, the method of transduction includes spinoculating, e.g., rotating or centrifuging, the transduction composition and optionally air in a centrifuge chamber for more than or about 5 minutes, e.g., more than or about 10 minutes, more than or about 15 minutes, more than or about 20 minutes, more than or about 30 minutes, more than or about 45 minutes, more than or about 60 minutes, more than or about 90 minutes, or more than or about 120 minutes. In some embodiments, the transduction composition and optionally air are rotated or centrifuged in a centrifuge chamber for more than 5 minutes but not more than 60 minutes, not more than 45 minutes, not more than 30 minutes, or not more than 15 minutes. In certain embodiments, the transduction includes rotating or centrifuging for 60 minutes or about 60 minutes.

In some embodiments, the method of transduction comprises injecting the transduction composition and optionally air into the centrifuge chamber for 10 to 60 minutes, 15 to 60 minutes, 15 to 45 minutes, 30 to 60 minutes, or 45 to 60 minutes, or about 10 to 60 minutes, about 15 to 60 minutes, about 15 to 45 minutes, about 30 to 60 minutes, or about 45 to 60 minutes, inclusive, and at 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 1900 g, 2000 g, 2100 g, 2200 g, 2300 g, 2400 g, 2500 g, 2600 g, 2700 g, 2800 g, 2900 g, 3000 g, 3100 g, 3200 g, 3300 g, 3400 g, 3500 g, 3600 g, 3700 g, 3800 g, 3900 g, 4000 g, 4100 g, 4200 g, 4300 g, 4400 g, 4500 g, 4600 g, 4700 g, 4800 g, 4900 g, 5000 g, 5100 g, 5200 g, 5300 g, 5400 g, 5500 g, 5600 g, 5700 g, 5800 g, 5900 g, 6000 g, 6100 g, 6200 g, 6300 g, 6400 g, 6500 g, g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g or 3600 g, about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g or 3600 g, or about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g or 3600 g. In certain embodiments, the method of transduction includes spinning or centrifuging the transduction composition, e.g., cells and viral vector particles, at or about 1600 g for 60 minutes or about 60 minutes.

In some embodiments, the transduction method does not include spinning or centrifugation.

2. Viral Vector Particles In some embodiments, the recombinant nucleic acid is delivered to the cell using a recombinant infectious viral particle, such as a vector derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is delivered to the T cell using a recombinant lentiviral or retroviral vector, such as a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557).

In some embodiments, retroviral vectors, such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV) or adeno-associated virus (AAV), have long terminal repeats (LTRs). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that they are capable of infecting several host cells, including humans. In one embodiment, the gene to be expressed replaces the gag, pol and/or env sequences of the retrovirus. A number of exemplary retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) Bio Techniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

Viral vector genomes are typically constructed in the form of a plasmid that can be transfected into a packaging or producer cell line. In any such instance, a nucleic acid encoding a recombinant protein, such as a recombinant receptor, is inserted or placed into a region of the viral vector, typically a non-essential region of the viral genome. In some embodiments, the nucleic acid is inserted into the viral genome in place of certain viral sequences to produce a virus that is replication-deficient.

Any of a wide variety of known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome. In some embodiments, at least two components are involved in the creation of a viral-based gene delivery system: first, a packaging plasmid that contains the structural proteins and enzymes necessary to generate viral vector particles, and second, the viral vector itself, i.e., the genetic material to be transferred. Biosafety safeguards can be incorporated into the design of one or both of these components.

In some embodiments, the packaging plasmid can contain any retroviral protein, such as HIV-1, other than the envelope protein (Naldini et al., 1998). In other embodiments, the viral vector can lack additional viral genes, such as those associated with pathogenicity, e.g., the primary transactivators of HIV, vpr, vif, vpu and nef, and/or Tat. In some embodiments, the lentiviral vector, such as an HIV-based lentiviral vector, contains only three genes of the parent virus, gag, pol and rev, which reduces or eliminates the possibility of reconstitution of wild-type virus by recombination.

In some embodiments, the viral vector genome is introduced into a packaging cell line that contains all the components necessary to package the viral genomic RNA transcribed from the viral vector genome into viral particles. Alternatively, the viral vector genome may contain one or more genes encoding viral components in addition to one or more sequences of interest, e.g., recombinant nucleic acids. However, in some aspects, endogenous viral genes required for replication are removed and provided separately to the packaging cell line to prevent replication of the genome in the target cell.

In some embodiments, the packaging cell line is transfected with one or more plasmid vectors containing the components necessary to generate the particles. In some embodiments, the packaging cell line is transfected with a plasmid containing the viral vector genome, including the LTR, cis-acting packaging sequences, and a nucleic acid encoding a sequence of interest, i.e., an antigen receptor such as CAR; and one or more helper plasmids encoding the enzymatic and/or structural components of the virus, such as Gag, pol, and/or rev. In some embodiments, multiple vectors are utilized to separate the various genetic components that generate the retroviral vector particles. In some such embodiments, providing the packaging cells with separate vectors reduces the chance of recombination events that might otherwise generate replication-competent virus. In some embodiments, a single plasmid vector carrying all retroviral components can be used.

In some embodiments, retroviral vector particles, such as lentiviral vector particles, are pseudotyped to increase the efficiency of transduction of host cells. For example, in some embodiments, retroviral vector particles, such as lentiviral vector particles, are pseudotyped with VSV-G glycoprotein, which provides a broad cellular host range and expands the cell types that can be transduced. In some embodiments, packaging cell lines are transfected with a plasmid or polynucleotide encoding a non-native envelope glycoprotein, e.g., to include a xenotropic, polytropic or amphotropic envelope, such as the Sindbis virus envelope, GALV or VSV-G.

In some embodiments, the packaging cell line provides components, including viral regulatory and structural proteins, required in trans to package viral genomic RNA into lentiviral vector particles. In some embodiments, the packaging cell line can be any cell line capable of expressing lentiviral proteins to produce functional lentiviral vector particles. In some aspects, suitable packaging cell lines include 293 cells (ATCC CCL X), 293T cells, HeLA cells (ATCC CCL 2), D17 cells (ATCC CCL 183), MDCK cells (ATCC CCL 34), BHK cells (ATCC CCL-10), and Cf2Th cells (ATCC CRL 1430).

In some embodiments, the packaging cell line stably expresses viral proteins. For example, in some aspects, a packaging cell line can be constructed that contains gag, pol, rev and/or other structural genes, but does not contain the LTRs and packaging components. In some embodiments, the packaging cell line can be transiently transfected with a nucleic acid molecule encoding one or more viral proteins along with a viral vector genome that contains a nucleic acid molecule encoding a heterologous protein and/or a nucleic acid encoding an envelope glycoprotein.

In some embodiments, the viral vector and packaging and/or helper plasmids are introduced into a packaging cell line via transfection or infection. The packaging cell line produces viral vector particles containing the viral vector genome. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection.

When the recombinant plasmid and retroviral LTRs and packaging sequences are introduced into a special cell line (e.g., by calcium phosphate precipitation), the packaging sequences can allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles that can then be secreted into the culture medium. In some embodiments, the medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. For example, in some aspects, after co-transfection of the packaging plasmid and transfer vector into a packaging cell line, viral vector particles are recovered from the culture medium and titrated by standard methods used by those of skill in the art.

In some embodiments, retroviral vectors, such as lentiviral vectors, can be produced in a packaging cell line, such as the exemplary HEK 293T cell line, by introduction of a plasmid that allows for the generation of lentiviral particles. In some embodiments, the packaging cells are transfected with and/or contain polynucleotides encoding gag and pol, as well as with and/or contain a polynucleotide encoding a recombinant receptor, such as an antigen receptor (e.g., CAR). In some embodiments, the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a rev protein. In some embodiments, the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a non-native envelope glycoprotein, such as VSV-G. In some such embodiments, about 2 days after transfection of the cells, e.g., HEK 293T cells, the cell supernatant contains the recombinant lentiviral vector, which can be harvested and titrated.

The recovered and/or produced retroviral vector particles can be used to transduce target cells using methods as described. Once inside the target cell, the viral RNA is reverse transcribed, transported to the nucleus, and stably integrated into the host genome. One or two days after integration of the viral RNA, expression of the recombinant protein, e.g., an antigen receptor such as a CAR, can be detected.

3. Incubation of cells In certain embodiments, genetic engineering, such as by transforming (e.g., transducing) a cell (e.g., an output composition) with a viral vector, further comprises one or more steps of incubating the cell after introducing or contacting the viral vector into the cell. In some embodiments, the cell, e.g., the cell of a transformed cell population, is incubated following the process of genetic engineering, transforming, transducing or transfecting the cell to introduce the viral vector into the cell. In certain embodiments, the incubation results in a population of incubated cells (also referred to herein as an incubated cell population).

In some embodiments, the cells are incubated after the introduction of the heterologous or recombinant polynucleotide, e.g., viral vector particle, is performed without further processing of the cells. In certain embodiments, prior to incubation, the cells are washed to remove or substantially remove exogenous or residual polynucleotides, e.g., encoding heterologous or recombinant polynucleotides, e.g., viral vector particles, e.g., that remain in the medium after the genetic engineering process following spinoculation.

In some such embodiments, further incubation is accomplished under conditions that result in integration of the viral vector into the host genome of one or more cells. It is within the level of ordinary skill in the art to assess or determine whether incubation has resulted in integration of the viral vector particles into the host genome, and thus empirically determine the conditions for further incubation. In some embodiments, integration of the viral vector into the host genome can be assessed by measuring the expression level of a recombinant protein, such as a heterologous protein, encoded by a nucleic acid contained in the genome of the viral vector particle after incubation. Numerous well-known methods for assessing the expression level of a recombinant molecule may be used, such as detection by affinity-based methods, such as immunoaffinity-based methods (e.g., in the context of cell surface proteins, such as by flow cytometry). In some examples, expression is measured by detection of a transduction marker and/or a reporter construct. In some embodiments, a nucleic acid encoding a truncated surface protein is included in the vector and used as a marker of expression and/or its enhancement.

In certain embodiments, the incubation is performed under static conditions, e.g., without centrifugation, shaking, rotation, rocking, or perfusion, e.g., continuous or semi-continuous perfusion, of the medium. In some embodiments, before or shortly after the start of the incubation, e.g., within 5 minutes, 15 minutes, or 30 minutes, the cells are transferred (e.g., transferred under sterile conditions) into a container such as a bag or vial and placed into an incubator.

In some embodiments, at least a portion of the incubation occurs within the internal cavity of a centrifuge chamber as described in International Publication No. WO2016/073602.

In some embodiments, cells into which a polynucleotide, e.g., a viral vector, encoding a heterologous or recombinant polypeptide has been introduced are transferred to a container for incubation. In some embodiments, the container is a vial. In certain embodiments, the container is a bag. In some embodiments, the cells, and optionally the heterologous or recombinant polypeptide, are transferred to the container under closed or sterile conditions. In some embodiments, the container, e.g., a vial or bag, is then placed in an incubator for all or part of the incubation. In certain embodiments, the incubator is set at 16°C, 24°C, or 35°C, about 16°C, 24°C, or 35°C, or at least 16°C, 24°C, or 35°C. In some embodiments, the incubator is set at 37°C, about 37°C, or 37°C ±2°C, ±1°C, ±0.5°C, or ±0.1°C.

In some aspects, conditions for incubation can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and/or stimuli such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

In some embodiments, the incubation is performed in serum-free medium. In some embodiments, the serum-free medium is a defined and/or precisely defined cell culture medium. In certain embodiments, the serum-free medium is a controlled culture medium that has been processed, e.g., filtered to remove inhibitors and/or growth factors. In some embodiments, the serum-free medium contains proteins. In certain embodiments, the serum-free medium may contain serum albumin, hydrolysates, growth factors, hormones, carrier proteins and/or attachment factors.

In certain embodiments, the cells are incubated in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In certain embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind and/or are capable of binding to a receptor expressed by and/or endogenous to the T cell. In certain embodiments, the one or more cytokines are or include members of the four-alpha-helical bundle family of cytokines. In some embodiments, members of the four-alpha-helical bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In certain embodiments, the one or more cytokines are or include IL-7. In certain embodiments, the one or more cytokines are or include recombinant IL-2.

In certain embodiments, the cells are incubated in the presence of IL-2, IL-7 and/or IL-15. In certain embodiments, the IL-2, IL-7 and/or IL-15 are recombinant. In certain embodiments, the IL-2, IL-7 and/or IL-15 are human. In certain embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7 and/or IL-15. In certain embodiments, the cells are incubated in the presence of recombinant IL-2, IL-7 and IL-15.

In some embodiments, cells, e.g., transformed cells, are incubated with a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 IU/mL and 1,000 IU/mL, between 10 IU/mL and 50 IU/mL, between 50 IU/mL and 100 IU/mL, between 100 IU/mL and 200 IU/mL, between 100 IU/mL and 500 IU/mL, between 250 IU/mL and 500 IU/mL, or between 500 IU/mL and 1,000 IU/mL.

In some embodiments, cells, e.g., transformed cells, are incubated with IL-2, e.g., human recombinant IL-2, at a concentration of between 1 IU/mL and 500 IU/mL, between 10 IU/mL and 250 IU/mL, between 50 IU/mL and 200 IU/mL, between 50 IU/mL and 150 IU/mL, between 75 IU/mL and 125 IU/mL, between 100 IU/mL and 200 IU/mL, or between 10 IU/mL and 100 IU/mL. In certain embodiments, the cells, e.g., transformed cells, are administered 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 100 IU/mL, or with recombinant IL-2 at a concentration of about 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 100 IU/mL. In some embodiments, the cells, e.g., transformed cells, are incubated in the presence of 100 IU/mL or about 100 IU/mL of recombinant IL-2, e.g., human recombinant IL-2.

In some embodiments, cells, e.g., transformed cells, are incubated with recombinant IL-7, e.g., human recombinant IL-7, at a concentration of between 100 IU/mL and 2,000 IU/mL, between 500 IU/mL and 1,000 IU/mL, between 100 IU/mL and 500 IU/mL, between 500 IU/mL and 750 IU/mL, between 750 IU/mL and 1,000 IU/mL, or between 550 IU/mL and 650 IU/mL. In certain embodiments, the cells, e.g., transformed cells, are administered 50 IU/mL, 100 IU/mL, 150 IU/mL, 200 IU/mL, 250 IU/mL, 300 IU/mL, 350 IU/mL, 400 IU/mL, 450 IU/mL, 500 IU/mL, 550 IU/mL, 600 IU/mL, 650 IU/mL, 700 IU/mL, 750 IU/mL, 800 IU/mL, 750 IU/mL, 750 IU/mL, 750 IU/mL, or 1,000 IU/mL. In certain embodiments, the cells, e.g., transformed cells, are incubated with IL-7 at a concentration of 600 IU/mL or about 600 IU/mL.

In some embodiments, cells, e.g., transformed cells, are incubated with recombinant IL-15, e.g., human recombinant IL-15, at a concentration of between 1 IU/mL and 500 IU/mL, between 10 IU/mL and 250 IU/mL, between 50 IU/mL and 200 IU/mL, between 50 IU/mL and 150 IU/mL, between 75 IU/mL and 125 IU/mL, between 100 IU/mL and 200 IU/mL, or between 10 IU/mL and 100 IU/mL. In certain embodiments, the cells, e.g., transformed cells, are administered 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL, or 200 IU/mL, or with recombinant IL-15 at a concentration of about 50 IU/mL, 60 IU/mL, 70 IU/mL, 80 IU/mL, 90 IU/mL, 100 IU/mL, 110 IU/mL, 120 IU/mL, 130 IU/mL, 140 IU/mL, 150 IU/mL, 160 IU/mL, 170 IU/mL, 180 IU/mL, 190 IU/mL or 200 IU/mL. In some embodiments, the cells, e.g., transformed cells, are incubated in the presence of 100 IU/mL or about 100 IU/mL of recombinant IL-15, e.g., human recombinant IL-2.

In certain embodiments, the cells, e.g., transformed cells, are incubated in the presence of IL-2, IL-7, and/or IL-15. In some embodiments, the IL-2, IL-7, and/or IL-15 are recombinant. In certain embodiments, the IL-2, IL-7, and/or IL-15 are human. In certain embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and/or IL-15. In certain embodiments, the cells are incubated in the presence of recombinant IL-2, IL-7, and IL-15.

In some embodiments, for example, all or part of the incubation for the non-expansion growth process is performed in a medium that includes basal medium (e.g., CTS OpTmizer Basal Medium (Thermofisher)), glutamine, and one or more recombinant cytokines, such as recombinant IL-2, IL-7, and/or IL-15. In some embodiments, the medium can contain one or more additional components as described in Section III.B. In some embodiments, the one or more additional components can include a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine). In some embodiments, the one or more additional components are provided by additional supplements, e.g., OpTmizer® supplements (Thermofisher). In some embodiments, the medium is serum-free medium and does not contain any serum components. In some aspects, the medium can contain one or more serum replacement proteins, such as one or more of albumin, insulin, or transferrin (e.g., CTS™ Immune Cell Serum Replacement). Exemplary serum-free media or components thereof are described in Section II.

In some embodiments, the cells are incubated in the presence of the same or similar medium that was present during stimulation of the cells, as in association with the stimulation methods or processes described above (e.g., on-column stimulation). In some embodiments, the cells are incubated in medium having the same cytokines as the medium that was present during stimulation of the cells, as in association with the stimulation methods or processes described above. In certain embodiments, the cells are incubated in medium having the same cytokines at the same concentrations as the medium that was present during stimulation of the cells, as in association with the stimulation methods or processes described above.

In some embodiments, the cells are incubated in the absence of recombinant cytokines. In some embodiments, the cells are incubated in the absence of one or more cytokines as described herein. In some embodiments, the cells are incubated in the absence of all of the cytokines described herein.

In some embodiments, all or part of the incubation is performed in a basal medium, e.g., a basal medium that does not contain one or more recombinant cytokines or does not contain any recombinant cytokines. In some embodiments, the medium does not contain one or more recombinant cytokines, e.g., recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15. In some aspects, the incubation is performed without any recombinant cytokines. In certain embodiments, the basal medium is supplemented with additional additives. In some embodiments, the basal medium is not supplemented with any additional additives. Additives to the cell culture medium can include, but are not limited to, nutrients, sugars, e.g., glucose, amino acids, vitamins, or additives such as ATP and NADH. Other additives can be added, but generally the specific additives and amounts are such that incubation of the medium with the cells promotes maintenance of the cells but does not minimize, limit, and/or induce metabolic activity of the cells during incubation.

In certain embodiments, the medium is a basal medium that does not contain one or more recombinant cytokines and does not contain serum components, i.e., serum-free medium, but may contain one or more additional components as described in Section III.B. In certain embodiments, the use of such serum-free medium, e.g., in all or part of the incubation of a non-expansion growth process, e.g., according to Section I-E-3, provides a lean medium that provides for the maintenance of the cells but does not contain certain factors that may promote the cells to be or likely to be in a resting or quiescent state by activating the cells or rendering them metabolically active. In some aspects, incubation in the presence of such serum-free medium (e.g., according to Section I-E-3) allows the cells to recover after stimulation and genetic manipulation (e.g., transduction). In some aspects, incubation in the presence of such serum-free medium (e.g., according to Section I-E-3) results in an output composition (e.g., therapeutic cell composition) containing cells that are less susceptible to loss or loss of viability, for example, during or after the manufacturing process, and when the harvested/formulated cells are cryopreserved and then thawed immediately prior to use. In some embodiments, the cells in the output composition (e.g., therapeutic cell composition) when thawed have lower levels of caspases or other apoptotic markers than cells incubated in a similar medium but containing one or more recombinant cytokines, serum or other factors that may render the cells more metabolically active upon cryopreservation of the output composition (e.g., therapeutic cell composition).

In some embodiments, the basal medium contains a mixture of inorganic salts, sugars, amino acids, and optionally vitamins, organic acids, and/or buffers or other well-known cell culture nutrients. In addition to nutrients, the medium also helps maintain pH and osmolality. In some aspects, the reagents of the basal medium support the growth, proliferation, and/or expansion of cells. A wide variety of commercially available basal media are known to those of skill in the art, including Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Iscove's Modified Dulbecco's Medium, and Ham's Medium. In some embodiments, the basal medium is Iscove's Modified Dulbecco's Medium, RPMI-1640, or α-MEM.

In some embodiments, the basal medium is a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS). In some embodiments, the basal medium is selected from Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), Alpha Minimum Essential Medium (AlphaMEM), Iscove's Modified Dulbecco's Medium, and M199. In some embodiments, the basal medium is a complex medium (e.g., RPMI-1640, IMDM). In some embodiments, the basal medium is OpTmizer™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher).

In some embodiments, the basal medium is protein-free. In some embodiments, the basal medium is free of human proteins (e.g., human serum proteins). In some embodiments, the basal medium is serum-free. In some embodiments, the basal medium is free of serum derived from a human. In some embodiments, the basal medium is free of recombinant proteins. In some embodiments, the basal medium is free of human proteins and recombinant proteins. In some embodiments, the basal medium is free of one or more or all of the cytokines as described herein.

In some embodiments, for example, all or part of the incubation of the non-expansion growth process is performed in basal medium without any additional additives or recombinant cytokines. In some embodiments, the basal medium is CTS OpTmizer basal medium (Thermofisher) without any additional additives or recombinant cytokines. In some embodiments, for example, all or part of the incubation of the non-expansion growth process is performed in a medium that includes basal medium and glutamine, for example, CTS OpTmizer basal medium with glutamine (Thermofisher).

In some embodiments, for example, all or part of the incubation of the non-expansion growth process is performed in a medium that includes a basal medium (e.g., CTS OpTmizer basal medium (Thermofisher)) that does not include one or more recombinant cytokines, e.g., recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15. In some embodiments, the medium is supplemented with one or more additional non-serum components as set forth in Section III.B. In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium further includes a free form of an amino acid, such as L-glutamine. In some embodiments, the serum-free medium does not include a serum replacement supplement. In some embodiments, the serum-free medium does not include a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine). In some embodiments, the serum-free medium does not include any recombinant cytokines. In some embodiments, the serum-free medium comprises a basal medium supplemented with a T cell supplement and a free form of L-glutamine, but does not contain any immune cell serum replacement, any dipeptide form of L-glutamine, or any recombinant cytokines. In some embodiments, the serum-free medium comprises a basal medium supplemented with a T cell supplement and a free form of L-glutamine, but does not contain any immune cell serum replacement, any dipeptide form of L-glutamine, or any recombinant cytokines. In some embodiments, the serum-free medium comprises a basal medium (e.g., supplemented OpTmizer™ T Cell Expansion Culture Basal Medium), L-glutamine, and one or more additional components, such as those provided by a supplement (e.g., OpTmizer™ T Cell Expansion Culture Supplement).

In particular embodiments, the cells ^are incubated in serum-free medium at a concentration of, or about, ^0.25 × ¹⁰⁶ cells/mL ^{, 0.5} ×106 cells/mL ^{, 0.75} ×106 cells/mL ^{, 1.0 ×} 106 cells/mL ^, 1.25×106 cells/mL, ^1.5 × ¹⁰⁶ cells/mL, 1.75× ¹⁰⁶ cells/mL or 2.0 ^{× 106} cells/mL. In particular embodiments, the cells are incubated in serum-free medium at a concentration of 0.25×10 ⁶ cells/mL to 1.0× ^{10 6} cells/mL or about 0.25×10 ⁶ cells/mL to 1.0×10 ⁶ cells/mL. In particular embodiments, the cells are incubated in serum-free medium at a concentration of 0.25×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL or about 0.25×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL. In particular embodiments, the cells are incubated in serum-free medium at a concentration of 0.5×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL or about 0.5×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL. In particular embodiments, the cells are incubated in serum-free medium at a concentration of 0.25×10 ⁶ cells/mL to 0.5×10 ⁶ cells/mL or about 0.25×10 ⁶ cells/mL to 0.5×10 ⁶ cells/mL. In particular embodiments, the cells are incubated in serum-free medium at a concentration of 0.75×10 ⁶ cells/mL or about 0.75×10 ⁶ cells/mL. In particular embodiments, the cells are incubated in serum-free medium at a concentration of 0.5×10 ⁶ cells/mL or about 0.5×10 ⁶ cells/mL. In some embodiments, the incubating step is for 18 hours to 30 hours or for about 18 hours to 30 hours. In particular embodiments, the incubating step is for 24 hours or about 24 hours or for 1 day or about 1 day.

In certain embodiments, the cells are incubated in the absence of cytokines. In certain embodiments, the cells are incubated in the absence of any recombinant cytokine. In certain embodiments, the cells are incubated in the absence of one or more recombinant cytokines, e.g., recombinant IL-2, IL-7, and/or IL-15.

In some embodiments, e.g., for non-expanded processes, all or a portion of the incubation is performed in a medium comprising basal medium, glutamine, and one or more recombinant cytokines, e.g., CTS OpTmizer basal medium (Thermofisher) containing glutamine and recombinant IL-2, IL-7, and/or IL-15. In some embodiments, e.g., for non-expanded processes, all or a portion of the incubation is performed in a medium comprising basal medium, glutamine, one or more recombinant cytokines, and T cell supplement, e.g., CTS OpTmizer basal medium (Thermofisher) containing glutamine, recombinant IL-2, IL-7, and/or IL-15, and OpTmizer® supplement (Thermofisher). In some embodiments, e.g., for non-expansion processes, all or a portion of the incubation is performed in a medium containing basal medium, glutamine, one or more recombinant cytokines, a T cell supplement, and one or more serum-substituting proteins, e.g., in CTS OpTmizer basal medium (Thermofisher) containing glutamine, recombinant IL-2, IL-7 and/or IL-15, OpTmizer® supplement (Thermofisher), and a serum-substituting protein such as albumin, insulin, or transferrin.

In some embodiments, the basal medium further comprises glutamine, e.g., L-glutamine. In some aspects, the glutamine is a free form of glutamine, e.g., L-glutamine. In some embodiments, the concentration of glutamine, e.g., L-glutamine, in the basal medium is about 0.5 mM to 1 mM, 0.5 mM to 1.5 mM, 0.5 mM to 2 mM, 0.5 mM to 2.5 mM, 0.5 mM to 3 mM, 0.5 mM to 3.5 mM, 0.5 mM to 4 mM, 0.5 mM to 4.5 mM, 0.5 mM to 5 mM, 1 mM to 1.5 mM, 1 mM to 2 mM, 1 mM to 2.5 mM, 1 mM to 3 mM, 1 mM to 3.5 mM, 1 mM to 4 mM, 1 mM to 4.5 mM, 1 mM to 5 mM, 1.5 mM to 2 mM, 1.5 mM to 2.5 mM, 1.5 mM to 3 mM, 1 .5mM to 3.5mM, 1.5mM to 4mM, 1.5mM to 4.5mM, 1.5mM to 5mM, 2mM to 2.5mM, 2mM to 3mM , 2mM to 3.5mM, 2mM to 4mM, 2mM to 4.5mM, 2mM to 5mM, 2.5mM to 3mM, 2.5mM to 3.5mM, 2. 5mM to 4mM, 2.5mM to 4.5mM, 2.5mM to 5mM, 3mM to 3.5mM, 3mM to 4mM, 3mM to 4.5mM, 3m M~5mM, 3.5mM~4mM, 3.5mM~4.5mM, 3.5mM~5mM, 4mM~4.5mM, 4mM~5mM or 4. 5mM to 5mM or about 0.5mM to 1mM, 0.5mM to 1.5mM, 0.5mM to 2mM, 0.5mM to 2.5mM, 0.5mM to 3mM, 0.5mM to 3.5mM, 0.5mM to 4mM, 0.5mM to 4.5mM, 0.5mM to 5mM, 1mM to 1. 5mM, 1mM to 2mM, 1mM to 2.5mM, 1mM to 3mM, 1mM to 3.5mM, 1mM to 4mM, 1mM to 4.5mM, 1m M~5mM, 1.5mM~2mM, 1.5mM~2.5mM, 1.5mM~3mM, 1.5mM~3.5mM, 1.5mM~4mM, 1 .5mM to 4.5mM, 1.5mM to 5mM, 2mM to 2.5mM, 2mM to 3mM, 2mM to 3.5mM, 2mM to 4mM, 2mM to 4.5mM, 2mM to 5mM, 2.5mM to 3mM, 2.5mM to 3.5mM, 2.5mM to 4mM, 2.5mM to 4.5mM, 2.5mM to 5mM, 3mM to 3.5mM, 3mM to 4mM, 3mM to 4.5mM, 3mM to 5mM, 3.5mM to 4mM, 3.5mM to 4.5mM, 3.5mM to 5mM, 4mM to 4.5mM, 4mM to 5mM or less than 4.5mM to 5mM (inclusive). In some embodiments, the concentration of glutamine, e.g., L-glutamine, in the basal medium is at least about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM. In some embodiments, the concentration of glutamine, e.g., L-glutamine, in the basal medium is up to about 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM. In some embodiments, the concentration of glutamine, e.g., L-glutamine, in the basal medium is about 2 mM.

In some embodiments, the basal medium may further comprise a protein or peptide. In some embodiments, the at least one protein is not of non-mammalian origin. In some embodiments, the at least one protein is human or derived from a human. In some embodiments, the at least one protein is recombinant. In some embodiments, the at least one protein comprises albumin, transferrin, insulin, fibronectin, aprotinin, or fetuin. In some embodiments, the protein comprises one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, insulin, or transferrin.

In some embodiments, the protein is albumin or an albumin substitute. In some embodiments, the albumin is human-derived albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is native human serum albumin. In some embodiments, the albumin is recombinant human serum albumin. In some embodiments, the albumin is recombinant albumin from a non-human source. The albumin substitute can be any protein or polypeptide source. Examples of such protein or polypeptide samples include, but are not limited to, bovine pituitary extract, plant hydrolysates (e.g., rice hydrolysates), bovine fetal albumin (fetuin), egg albumin, human serum albumin (HSA), or another animal-derived albumin, chicken extract, bovine embryo extract, AlbuMAX® I, and AlbuMAX® II. In some embodiments, the protein or peptide comprises transferrin. In some embodiments, the protein or peptide comprises fibronectin. In some embodiments, the protein or peptide comprises aprotinin. In some embodiments, the protein comprises fetuin.

In some embodiments, the one or more additional proteins are part of a serum replacement supplement that is added to the basal medium. Examples of serum replacement supplements include, for example, Immune Cell Serum Replacement (ThermoFisher, #A2598101) or those described in Smith et al. Clin Transl Immunology. 2015 Jan;4(1):e31.

In certain embodiments, the cells are incubated for about 18, 24, 30, 36, 40, 48, 54, 60, 72, 84, 96 or more than 96 hours, or for at least 18, 24, 30, 36, 40, 48, 54, 60, 72, 84, 96 or more than 96 hours, after introduction of a polynucleotide encoding a heterologous or recombinant protein, e.g., a viral vector. In certain embodiments, the cells are incubated for 1, 2, 3, 4 or more days, about 1, 2, 3, 4 or more days, or at least 1, 2, 3, 4 or more days after introduction of a polynucleotide encoding a heterologous or recombinant protein, e.g., a viral vector. In some embodiments, the incubating is performed for a time period between 30 minutes and 2 hours, between 1 hour and 8 hours, between 6 hours and 12 hours, between 12 hours and 18 hours, between 16 hours and 24 hours, between 18 hours and 30 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours, between 96 hours and 120 hours, between 90 hours, between 1 day and 7 days, between 3 days and 8 days, between 1 day and 3 days, between 4 days and 6 days, or between 4 days and 5 days prior to genetic manipulation. In some embodiments, the incubating is for at or about 18 to 30 hours. In certain embodiments, the incubating is for at or about 24 hours or for at or about 1 day.

In certain embodiments, the total incubation period is 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours or 120 hours, about 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours or 120 hours, or at least 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours or 120 hours. In certain embodiments, the total incubation period is 1 day, 2 days, 3 days, 4 days or 5 days, about 1 day, 2 days, 3 days, 4 days or 5 days, or at least 1 day, 2 days, 3 days, 4 days or 5 days. In certain embodiments, the incubation is completed within about 120, 108, 96, 84, 72, 60, 54, 48, 42, 36, 30, 24, 18, or 12 hours, or within 120, 108, 96, 84, 72, 60, 54, 48, 42, 36, 30, 24, 18, or 12 hours. In certain embodiments, incubation is completed within 1, 2, 3, 4, or 5 days, about 1, 2, 3, 4, or 5 days, or within 1, 2, 3, 4, or 5 days. In some embodiments, the total incubation period is 12 to 120 hours, 18 to 96 hours, 24 to 72 hours, or 24 to 48 hours, or about 12 to 120 hours, 18 to 96 hours, 24 to 72 hours, or 24 to 48 hours, inclusive. In some embodiments, the total incubation period is 1 to 48 hours, 4 to 36 hours, 8 to 30 hours, or 12 to 24 hours, inclusive, or about 1 to 48 hours, 4 to 36 hours, 8 to 30 hours, or 12 to 24 hours, inclusive. In certain embodiments, the incubation is carried out for at or about 24, 48 or 72 hours, or at or about 1, 2 or 3 days, respectively. In certain embodiments, the incubation is carried out for at or about 24 hours ±6 hours, 48 hours ±6 hours, or 72 hours ±6 hours. In certain embodiments, the incubation is carried out for at or about 72 hours, or at or about 3 days. In some embodiments, the incubation is carried out for a period sufficient to allow integration of the polynucleotide encoding the heterologous or recombinant protein into the genome of the cell.

In certain embodiments, incubation is initiated at about, or at least 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours after the start of stimulation. In certain embodiments, incubation is initiated at about, or at least 0.5 days, 1 day, 1.5 days, or 2 days after the start of stimulation. In certain embodiments, incubation is initiated within about 120, 108, 96, 84, 72, 60, 54, 48, 42, 36, 30, 24, 18, or 12 hours, or within 120, 108, 96, 84, 72, 60, 54, 48, 42, 36, 30, 24, 18, or 12 hours of the start of stimulation. In certain embodiments, incubation is initiated 11, 10, 9, 8, 7, 6, 5, or 4 hours after the start of stimulation, about 11, 10, 9, 8, 7, 6, 5, or 4 hours after the start of stimulation, within 11, 10, 9, 8, 7, 6, 5, or 4 hours after the start of stimulation. In certain embodiments, incubation is initiated 5, 4, 3, 2, 1, or 0.5 days, about 5, 4, 3, 2, 1, or 0.5 days, or within 5, 4, 3, 2, 1, or 0.5 days after the start of stimulation.

In some embodiments, the incubation is completed 24 to 120 hours, 36 to 108 hours, 48 to 96 hours, or 48 to 72 hours, inclusive, after the start of stimulation. In some embodiments, the incubation is completed within 120 hours, 108 hours, 96 hours, 72 hours, 48 hours, or 36 hours, about 120 hours, 108 hours, 96 hours, 72 hours, 48 hours, or 36 hours, or within 120 hours, 108 hours, 96 hours, 72 hours, 48 hours, or 36 hours, inclusive, after the start of stimulation. In some embodiments, the incubation is completed within 5, 4.5, 4, 3, 2, or 1.5 days, about 5, 4.5, 4, 3, 2, or 1.5 days, or within 5, 4.5, 4, 3, 2, or 1.5 days from the start of stimulation. In certain embodiments, the incubation is completed after 24 hours ± 6 hours, 48 hours ± 6 hours, or 72 hours ± 6 hours after the start of stimulation. In some embodiments, the incubation is completed after 72 hours or about 72 hours, or after 3 days or about 3 days.

In some of the above embodiments, the engineered cells are not incubated under culture conditions to expand the cell population (e.g., number of viable T cells). In some of the above embodiments, the cells are not incubated under culture conditions that increase the amount of viable cells during incubation or culture. For example, in some aspects, the cells are not incubated under conditions (e.g., culture conditions) that increase the amount of total viable cells at the end of incubation compared to the number of total viable cells at the start of incubation. In some embodiments, the cells are incubated under conditions that may result in expansion, but the incubation conditions are not performed with the intent of expanding the cell population. In some embodiments, cells incubated under conditions that do not promote or encourage expansion and proliferation may be referred to as non-expanded or minimally expanded cells (see Sections I-G).

In some embodiments, the transduced or engineered cells are incubated under culture conditions that promote growth and/or expansion following a genetic engineering step, such as introducing a recombinant polypeptide into the cells by transduction or transfection. In certain embodiments, the cells are cultured after the cells are transduced or transfected with a recombinant polynucleotide, such as a polynucleotide encoding a recombinant receptor. In some embodiments, the culture produces one or more culture compositions of engineered T cells. In some embodiments, such culture conditions can be designed to induce proliferation, expansion, activation and/or survival of cells in the population. In certain embodiments, the culture conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to promote cell growth, division and/or expansion. In some embodiments, cells incubated under conditions that promote proliferation and/or expansion may be referred to as expanded cells (see Sections I-G).

In particular embodiments, the cells are incubated (e.g. ^, cultured) under culture conditions at a concentration of, or about, ^0.25 × ¹⁰⁶ cells/mL ^{, 0.5} × ¹⁰⁶ cells/mL, 0.75× ¹⁰⁶ cells/mL, 1.0× ¹⁰⁶ cells ^/ mL ^, 1.25× ¹⁰⁶ cells/mL ^{, 1.5} × ¹⁰⁶ cells/mL, 1.75× ¹⁰⁶ cells/mL, or 2.0× ¹⁰⁶ cells/mL. In particular embodiments, the cells are incubated under culture conditions at a concentration of 0.25×10 ⁶ cells/mL to 1.0×10 ⁶ cells/mL or about 0.25×10 ⁶ cells/mL to 1.0×10 ⁶ cells/mL. In particular embodiments, the cells are incubated under culture conditions at a concentration of 0.25×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL or about 0.25×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL. In particular embodiments, the cells are incubated under culture conditions at a concentration of 0.5×10 ⁶ cells/mL to 0.75×10 ⁶ cells/mL or about 0.5×10 ⁶ cells/mL to 0.75× ^{10 6} cells/mL. In particular embodiments, the cells are incubated under culture conditions at a concentration of 0.25× ¹⁰⁶ cells/mL to 0.5× ¹⁰⁶ cells/mL or about 0.25× ¹⁰⁶ cells/mL to 0.5× ¹⁰⁶ cells/mL. In particular embodiments, the cells are incubated under culture conditions at a concentration of 0.75× ¹⁰⁶ cells/mL or about 0.75× ¹⁰⁶ cells/mL. In particular embodiments, the cells are incubated under culture conditions at a concentration of 0.5× ¹⁰⁶ cells/mL or about 0.5× ¹⁰⁶ cells/mL.

In some embodiments, the engineered cells are cultivated (e.g., cultured) in a vessel that can be filled with cell medium and/or cells, e.g., through an inlet, for culturing the added cells. The cells can be from any cell source where culturing cells is desired, e.g., for cell expansion and/or proliferation.

In some aspects, the culture medium is a culture medium adapted to support the growth, expansion or proliferation of cells, such as T cells. In some aspects, the medium can be a liquid containing a mixture of salts, amino acids, vitamins, sugars, or any combination thereof. In some embodiments, the culture medium further contains one or more stimulatory conditions or stimuli, for example, to stimulate the expansion or proliferation of the cells during incubation. In some embodiments, the stimulatory conditions are or include one or more cytokines, such as selected from IL-2, IL-7, or IL-15. In some embodiments, the cytokine is a recombinant cytokine. In particular embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines have the ability to bind and/or bind to a receptor expressed by and/or endogenous to the T cell. In particular embodiments, the one or more cytokines are or include members of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the four-alpha helix bundle cytokine family include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more cytokines are or include IL-15. In particular embodiments, the one or more cytokines are or include IL-7. In particular embodiments, the one or more cytokines are or include recombinant IL-2.

In some embodiments, the concentration of one or more cytokines in the culture medium during culture is, independently, between 1 IU/mL and 1500 IU/mL or between about 1 IU/mL and 1500 IU/mL, e.g., between 1 IU/mL and 100 IU/mL, between 2 IU/mL and 50 IU/mL, between 5 IU/mL and 10 IU/mL, between 10 IU/mL and 500 IU/mL, between 50 IU/mL and 250 IU/mL, or between 100 IU/mL and 200 IU/mL, between 50 IU/mL and 1500 IU/mL. , 100 IU/mL to 1000 IU/mL or 200 IU/mL to 600 IU/mL, or about 1 IU/mL to 100 IU/mL, 2 IU/mL to 50 IU/mL, 5 IU/mL to 10 IU/mL, 10 IU/mL to 500 IU/mL, 50 IU/mL to 250 IU/mL or 100 IU/mL to 200 IU/mL, 50 IU/mL to 1500 IU/mL, 100 IU/mL to 1000 IU/mL or 200 IU/mL to 600 IU/mL. In some embodiments, the concentration of one or more cytokines is, independently, at least 1 IU/mL, 5 IU/mL, 10 IU/mL, 50 IU/mL, 100 IU/mL, 200 IU/mL, 500 IU/mL, 1000 IU/mL, or 1500 IU/mL, or at least about 1 IU/mL, 5 IU/mL, 10 IU/mL, 50 IU/mL, 100 IU/mL, 200 IU/mL, 500 IU/mL, 1000 IU/mL, or 1500 IU/mL.

In some embodiments, the engineered cell composition is cultured at a temperature between 25-38°C, e.g., between 30-37°C, e.g., at or about 37°C±2°C. In some embodiments, the culture conditions are carried out for a period of time until the culture, e.g., cultivation or expansion, results in a desired or threshold density, concentration, number or dosage of cells. In some embodiments, incubation is carried out for a period of time until the culture, e.g., cultivation or expansion, results in a desired or threshold density, concentration, number or dosage of cells. In some embodiments, the incubation is for greater than or equal to about 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more, or for greater than or equal to about 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more, or for greater than or equal to about 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more, or for greater than or equal to about 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more.

In some embodiments, the cells are incubated or cultured under conditions that maintain a target amount of carbon dioxide in the cell culture. In some aspects, this ensures optimal culture, expansion and proliferation of the growing cells. In some aspects, the amount of carbon dioxide (CO ₂ ) is between 10% and 0% (v/v) of the gas, such as between 8% and 2% (v/v) of the gas, such as an amount of 5% or about 5% (v/v) CO ₂ .

In certain embodiments, the culturing is performed in a closed system. In certain embodiments, the culturing is performed in a closed system under sterile conditions. In some embodiments, the composition of engineered cells is removed from the closed system and placed into and/or connected to a bioreactor for culturing. Examples of suitable bioreactors for culturing include, but are not limited to, the GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20|50, Finesse SmartRocker bioreactor system, and Pall XRS bioreactor system. In some embodiments, the bioreactor is used to perfuse and/or mix the cells during at least a portion of the culturing process.

In some embodiments, cells cultured enclosed, connected, and/or controlled by a bioreactor expand more rapidly during culture than cells cultured without a bioreactor, e.g., cells cultured under static conditions, e.g., without mixing, agitation, motion, and/or perfusion. In some embodiments, cells cultured enclosed, connected, and/or controlled by a bioreactor reach or achieve a threshold expansion, cell number, and/or cell density within 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 60 hours, 48 hours, 36 hours, 24 hours, or 12 hours. In some embodiments, cells cultured while enclosed in, connected to, and/or under the control of a bioreactor reach or achieve a threshold expansion, cell number, and/or cell density at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold greater than cells cultured in exemplary and/or alternative processes in which the cells are not enclosed in, connected to, and/or under the control of a bioreactor.

In some embodiments, the mixing is or includes rocking and/or motioning. In some embodiments, the cells are incubated using a container, e.g., a bag, used in connection with the bioreactor. Optionally, the bioreactor can be subjected to vibration or rocking, which in some aspects can increase oxygen transfer. The motion of the bioreactor can include, but is not limited to, rotation along the horizontal axis of the bioreactor, rotation along a vertical axis, rocking motion along a tilted or inclined horizontal axis, or any combination thereof. In some embodiments, at least a portion of the incubation is performed with rocking. The rocking speed and rocking angle can be adjusted to achieve the desired agitation. In some embodiments, the rocking angle is at or about 20°, 19°, 18°, 17°, 16°, 15°, 14°, 13°, 12°, 11°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, or 1°. In certain embodiments, the rocking angle is 6-16°. In other embodiments, the rocking angle is 7-16°. In other embodiments, the rocking angle is 8-12°. In some embodiments, the rocking speed is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 rpm. In some embodiments, the rocking speed is 4 to 12 rpm, inclusive, e.g., 4 to 6 rpm. At least a portion of the cell culture expansion is performed with rocking motion, e.g., at an angle of 5° to 10°, e.g., 6°, at a constant rocking speed, e.g., 5 to 15 RPM, e.g., at a speed of 6 RPM or 10 RPM.

In some embodiments, a composition comprising cells, e.g., engineered cells, e.g., engineered T cells, engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells, is cultured in the presence of a detergent. In certain embodiments, culturing the cells of the composition reduces the amount of shear stress that may occur during culture, e.g., due to mixing, rocking, movement, and/or perfusion. In particular embodiments, a composition of cells, e.g., engineered cells, e.g., engineered T cells, engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells, are cultured with a detergent and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the T cells remain viable, e.g., survive and/or do not undergo necrosis, programmed cell death, or apoptosis during the culture or at least 1, 2, 3, 4, 5, 6, 7, or more than 7 days after completion of the culture. In certain embodiments, a composition of cells, e.g., engineered T cells, e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells, is cultured in the presence of a detergent, and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the cells undergo cell death, e.g., programmed cell death, apoptosis, and/or necrosis, e.g., due to shear or shear-induced stress.

In certain embodiments, the composition of cells, e.g., engineered T cells, e.g., engineered CD4+ T cells or engineered CD8+ T cells, is cultured in the presence of 0.1 μl/ml to 10.0 μl/ml, 0.2 μl/ml to 2.5 μl/ml, 0.5 μl/ml to 5 μl/ml, 1 μl/ml to 3 μl/ml, or 2 μl/ml to 4 μl/ml of a surfactant. In some embodiments, a composition of cells, e.g., engineered T cells, e.g., engineered CD4+ T cells or engineered CD8+ T cells, is at or about 0.1 μl/ml, 0.2 μl/ml, 0.4 μl/ml, 0.6 μl/ml, 0.8 μl/ml, 1 μl/ml, 1.5 μl/ml, 2.0 μl/ml, 2.5 μl/ml, 5.0 μl/ml, 10 μl/ml, 25 μl/ml or 50 μl/ml. The composition is cultured in the presence of 0.8 μl/ml, 1 μl/ml, 1.5 μl/ml, 2.0 μl/ml, 2.5 μl/ml, 5.0 μl/ml, 10 μl/ml, 25 μl/ml, or 50 μl/ml, or at least 0.1 μl/ml, 0.2 μl/ml, 0.4 μl/ml, 0.6 μl/ml, 0.8 μl/ml, 1 μl/ml, 1.5 μl/ml, 2.0 μl/ml, 2.5 μl/ml, 5.0 μl/ml, 10 μl/ml, 25 μl/ml, or 50 μl/ml of a surfactant. In certain embodiments, the composition of cells is cultured in the presence of 2 μl/ml or about 2 μl/ml of a surfactant.

In some embodiments, the surfactant is or includes an agent that reduces the surface tension of liquids and/or solids. For example, the surfactant can be a fatty alcohol (e.g., steryl alcohol), a polyoxyethylene glycol octylphenol ether (e.g., Triton X-100), or a polyoxyethylene glycol sorbitan alkyl ester (e.g., polysorbate 20, 40, 60). In certain embodiments, the surfactant is selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and poloxamer 188 (P188). In an exemplary embodiment, the concentration of the surfactant in the chemically defined feed media is about 0.0025% to about 0.25% (v/v) PS80, about 0.0025% to about 0.25% (v/v) PS20, or about 0.1% to about 5.0% (w/v) P188.

In some embodiments, the surfactant is or includes an anionic surfactant, cationic surfactant, zwitterionic surfactant, or nonionic surfactant added thereto. Suitable anionic surfactants include, but are not limited to, alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, sodium dodecyl sulfate, alkyl polyoxyethylene sulfate, sodium alginate, dioctyl sodium sulfosuccinate, phosphatidylglycerol, phosphatidylinosine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylserine, phosphatidic acid and salts thereof, sodium carboxymethylcellulose, cholic acid and other bile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid) and salts thereof (e.g., sodium deoxycholate).

In some embodiments, suitable nonionic surfactants include glyceryl esters, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters (polysorbates), polyoxyethylene fatty acid esters, sorbitan esters, glycerol monostearate, polyethylene glycol, polypropylene glycol, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxamers), poloxamines, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, noncrystalline cellulose, polysaccharides including starch and starch derivatives, such as hydroxyethyl starch (HES), polyvinyl alcohol, and polyvinylpyrrolidone. In certain embodiments, the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene, preferably a block copolymer of propylene glycol and ethylene glycol. Such polymers are sold under the trademark Poloxamer, and may also be referred to as PLURONIC® F68 or Kolliphor® P188. Polyoxyethylene fatty acid esters include those with short alkyl chains. One example of such a surfactant is SOLUTOL® HS15, polyethylene-660-hydroxystearate.

In some embodiments, suitable cationic surfactants include, but are not limited to, natural phospholipids, synthetic phospholipids, quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitosan, lauryldimethylbenzylammonium chloride, acylcarnitine hydrochloride, dimethyldioctadecylammonium bromide (DDAB), dioleyltrimethyl ammonium propane (DOTAP), dimyristoyltrimethylammonium propane (DMTAP), dimethylaminoethanecarbamoylcholesterol (DC-Chol), 1,2-diacylglycero-3-(O-alkyl)phosphocholines, O-alkylphosphatidylcholines, alkylpyridinium halides, or long chain alkylamines such as n-octylamine and oleylamine.

Zwitterionic surfactants are electrically neutral, but have localized positive and negative charges within the same molecule. Suitable zwitterionic surfactants include, but are not limited to, zwitterionic phospholipids. Suitable phospholipids include phosphatidylcholine, phosphatidylethanolamine, diacyl-glycero-phosphoethanolamine (e.g., dimyristoyl-glycero-phosphoethanolamine (DMPE), dipalmitoyl-glycero-phosphoethanolamine (DPPE), distearoyl-glycero-phosphoethanolamine (DSPE), and dioleolyl-glycero-phosphoethanolamine (DOPE)). The present invention may use mixtures of phospholipids, including anionic and zwitterionic phospholipids. Such mixtures include, but are not limited to, lysophospholipids, egg phospholipids, or soybean phospholipids, or any combination thereof. The phospholipids may be anionic, zwitterionic, or a mixture of phospholipids, salted or desalted, hydrogenated or partially hydrogenated, or natural, semi-synthetic, or synthetic.

In certain embodiments, the surfactant is a poloxamer, e.g., poloxamer 188. In some embodiments, the composition of cells is cultured in the presence of 0.1 μl/ml to 10.0 μl/ml, 0.2 μl/ml to 2.5 μl/ml, 0.5 μl/ml to 5 μl/ml, 1 μl/ml to 3 μl/ml, or 2 μl/ml to 4 μl/ml of poloxamer. In some embodiments, the composition of cells is at or about 0.1 μl/ml, 0.2 μl/ml, 0.4 μl/ml, 0.6 μl/ml, 0.8 μl/ml, 1 μl/ml, 1.5 μl/ml, 2.0 μl/ml, 2.5 μl/ml, 5.0 μl/ml, 10 μl/ml, 25 μl/ml or 50 μl/ml. The composition is cultured in the presence of at least 0.1 μl/ml, 0.2 μl/ml, 0.4 μl/ml, 0.6 μl/ml, 0.8 μl/ml, 1 μl/ml, 1.5 μl/ml, 2.0 μl/ml, 2.5 μl/ml, 5.0 μl/ml, 10 μl/ml, 25 μl/ml or 50 μl/ml of a surfactant. In certain embodiments, the composition of cells is cultured in the presence of 2 μl/ml or about 2 μl/ml of a poloxamer.

In some aspects, engineered T cell populations (e.g., CD4, CD8) can be expanded separately or together until each reaches a threshold mass or cell density. In certain embodiments, the culture is terminated, such as by harvesting the cells, when the cells reach a threshold mass, concentration and/or expansion. In certain embodiments, the culturing is terminated when the cells have achieved, or about, or at least, a 1.5-fold expansion, a 2-fold expansion, a 2.5-fold expansion, a 3-fold expansion, a 3.5-fold expansion, a 4-fold expansion, a 4.5-fold expansion, a 5-fold expansion, a 6-fold expansion, a 7-fold expansion, a 8-fold expansion, a 9-fold expansion, a 10-fold expansion, or more than 10-fold expansion, e.g., based on and/or compared to the amount of cell density at the initiation or start of the culturing. In some embodiments, the threshold expansion is, for example, a 4-fold expansion based on and/or relative to the amount of cell density at the initiation or start of the culture. In some embodiments, the culture is terminated, such as by harvesting the cells, when the cells achieve a threshold total cell amount, such as a threshold cell number. In some embodiments, the culture is terminated, such as by harvesting the cells, when the cells achieve a threshold total nucleated cell (TNC) number. In some embodiments, the culture is terminated, such as when the cells achieve a threshold cell viability, such as a threshold viable cell number. In some embodiments, the threshold cell number is 50× ¹⁰⁶ cells, 100× ¹⁰⁶ cells, 200× ¹⁰⁶ cells, 300× ¹⁰⁶ cells, 400× ¹⁰⁶ cells, 600×106 cells, 800× ¹⁰⁶ cells, 1000× ¹⁰⁶ cells, 1200× ¹⁰⁶ cells, 1400× ¹⁰⁶ cells, 1600× ¹⁰⁶ cells, 1800× ¹⁰⁶ cells, 2000× ¹⁰⁶ cells, 2500× ¹⁰⁶ cells, 3000× ¹⁰⁶ cells, 4000× ¹⁰⁶ cells, 5000× ¹⁰⁶ cells, 10,000× ¹⁰⁶ cells, 12,000× ¹⁰⁶ cells, 15,000× ¹⁰⁶ cells, or 20,000× ¹⁰⁶ cells, or about 50 ^× 10 ⁶ cells, 100×10 ⁶ cells, 200×10 ⁶ cells, 300×10 ⁶ cells, 400×10 ⁶ cells, 600×10 ⁶ cells, 800×10 ⁶ cells, 1000×10 ⁶ cells, 1200×10 ⁶ cells, 1400×10 ⁶ cells, 1600×10 ⁶ cells, 1800×10 ⁶ cells, 2000×10 ⁶ cells, 2500×10 ⁶ cells, 3000×10 ⁶ cells, 4000×10 ⁶ cells, 5000×10 ^{6 cells, 10,000×10 6} cells, 12,000×10 ⁶ cells, 15,000×10 ⁶ cells or 20,000×10 ⁶ cells, or at least 50×10 ⁶ cells, 100 ^× 10 ⁶ cells, 200× ¹⁰⁶ cells, 300× ¹⁰⁶ cells, 400× ¹⁰⁶ cells, 600× ¹⁰⁶ cells, 800× ¹⁰⁶ cells, 1000× ¹⁰⁶ cells, 1200×106 cells, 1400× ¹⁰⁶ cells, 1600× ¹⁰⁶ cells, 1800× ¹⁰⁶ cells, 2000× ¹⁰⁶ cells, 2500× ¹⁰⁶ cells, 3000× ¹⁰⁶ cells, 4000× ¹⁰⁶ cells, 5000× ¹⁰⁶ cells, 10,000× ¹⁰⁶ cells, 12,000× ¹⁰⁶ cells, 15,000× ¹⁰⁶ cells or 20,000× ¹⁰⁶ cells, or viable cells within any of ^the above thresholds.

In certain embodiments, culture is terminated when cells reach threshold cell number.In some embodiments, culture is terminated 6 hours, 12 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more, or within about 6 hours, 12 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more, or within 6 hours, 12 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more, or within 1 day or about 1 day after threshold cell number is reached.In certain embodiments, culture is terminated 1 day or about 1 day after threshold cell number is reached. In certain embodiments, the threshold density is 0.1 x ¹⁰⁶ cells/ ^ml , ^{0.5 x} 106 cells/ml, 1 x 106 cells/ml, ^1.2 x ¹⁰⁶ cells/ml, ^1.5 x 106 cells/ml ^{, 1.6 x 106} cells/ml, 1.8 ^x 106 cells/ml, 2.0 x 106 cells/ml, ^2.5 x 106 cells/ml, 3.0 x 106 cells/ml, ^3.5 x 106 cells/ml, ^4.0 x 106 cells/ml, 4.5 x 106 cells/ml, 5.0 x 106 cells/ml, 6 x ¹⁰⁶ cells/ml, 8 x ¹⁰⁶ cells/ml or 10 x ¹⁰⁶ cells/ml, or about 0.1 x ¹⁰⁶ cells/ml, ^{0.5 x 106} cells/ml, 1 x 10 ⁶ cells/ ^ml , ^{1.2x106 cells} /ml, 1.5x106 ^cells /ml, ^1.6x106 cells/ml, ^1.8x106 cells/ml, ^2.0x106 cells/ml, ^2.5x106 cells/ml, ^3.0x106 cells/ml, 3.5x106 cells/ml, ^4.0x106 cells/ml, ^4.5x106 cells/ml, ^5.0x106 cells/ml, ^6x106 cells/ml, 8x106 cells/ml or ^10x106 cells/ml or at least ^0.1x106 cells/ml, ^0.5x106 cells/ml, ^1x106 cells/ml, ^1.2x106 cells/ml, ^1.5x106 cells/ml, 1.6x10 ⁶ cells/ml, 1.8x106 cells/ml, ^2.0x106 cells/ml, ^2.5x106 cells/ml ^{, 3.0x106} cells/ml ^{, 3.5x106 cells} /ml, ^4.0x106 cells/ml, ^4.5x106 cells/ml, 5.0x106 cells/ml, 6x106 cells/ml, ^8x106 cells/ml or ^10x106 cells/ml, or any of the above thresholds of viable cells. In certain embodiments, the culture is terminated when the cells achieve the threshold density. In some embodiments, the culture is terminated at or within 6 hours, 12 hours, 24 hours, 36 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or more than 7 days after the threshold density is reached. In certain embodiments, the culture is terminated at or about 1 day after the threshold density is reached.

In some embodiments, at least a portion of the culture is performed under static conditions. In some embodiments, at least a portion of the culture is performed with perfusion, e.g., to flush out spent medium and flush in fresh medium during culture. In some embodiments, the method includes perfusing fresh culture medium into the cell culture, e.g., through a feed port. In some embodiments, the culture medium added during perfusion contains one or more stimuli, e.g., one or more recombinant cytokines, e.g., IL-2, IL-7, and/or IL-15. In some embodiments, the culture medium added during perfusion is the same culture medium used during static incubation.

In some embodiments, following incubation, the container, e.g., bag, is reconnected to a system for performing one or more other processing steps for manufacturing, producing, or creating the cellular therapeutic, e.g., a system containing a centrifuge chamber. In some aspects, the cultured cells are transferred from the bag to the interior cavity of the chamber to formulate the cultured cells.

a. Monitoring of cells during incubation In some embodiments, cells are monitored during the incubation step, e.g., under expansion (e.g., culture) or under low/no expansion (e.g., incubation). Monitoring can be performed, for example, to confirm (e.g., measure, quantify) cell morphology, cell phenotype, cell viability, cell death, and/or cell concentration (e.g., viable cell concentration). In some embodiments, monitoring is performed manually, e.g., by a human operator. In some embodiments, monitoring is performed by an automated system. An automated system requires little or no manual input to the cultured cells. In some embodiments, monitoring is performed manually or by an automated system.

In certain embodiments, the cells are monitored by an automated system that does not require any manual input. In some embodiments, the automated system is adapted to a bioreactor, such as a bioreactor described herein, or an incubator, such as those described herein, such that cells undergoing incubation, e.g., under expansion or low expansion conditions, can be removed from the bioreactor or incubator, monitored, and then returned to the bioreactor or incubator. In some embodiments, the monitoring and incubation are performed in a closed loop configuration. In some aspects, in a closed loop configuration, the automated system and the bioreactor or incubator remain sterile. In embodiments, the automated system is sterile. In some embodiments, the automated system is an in-line system.

In some embodiments, the automated system includes the use of optical techniques (e.g., microscopy) to detect cell morphology, cell phenotype, cell viability, cell death, and/or cell concentration (e.g., viable cell concentration). Any optical technique suitable for determining cell attributes, viability, and concentration, etc. is contemplated herein. Non-limiting examples of useful optical techniques include bright field microscopy, fluorescence microscopy, differential interference contrast (DIC) microscopy, phase contrast microscopy, digital holography microscopy (DHM), differential digital holography microscopy (DDHM), or combinations thereof. Differential digital holography microscopy, DDHM, and differential DHM are used interchangeably herein. In certain embodiments, the automated system includes a differential digital holography microscope. In certain embodiments, the automated system includes a differential digital holography microscope including an illumination means (e.g., laser, led). Descriptions and uses of DDHM methods can be found, for example, in US 7,362,449, EP 1,631,788, US 9,904,248, and US 9,684,281, which are incorporated by reference in their entireties.

DDHM allows label-free non-destructive imaging of cells, resulting in high-contrast holographic images. The images may be subjected to object segmentation and further analysis to obtain multiple morphological features that quantitatively describe the imaged object (e.g., cultured cells, cell debris). Thus, various features (e.g., cell morphology, phenotype, cell viability, cell concentration) may be directly assessed or calculated from the DDHM, for example, using steps of image acquisition, image processing, image segmentation, and feature extraction. In some embodiments, the automated system includes a digital recording device for recording the holographic image. In some embodiments, the automated system includes a computer that includes an algorithm for analyzing the holographic image. In some embodiments, the automated system includes a monitor and/or computer for displaying the results of the holographic image analysis. In some embodiments, the analysis is automated (i.e., can be performed without user input). Examples of suitable automated systems for monitoring cells during the culture process include, but are not limited to, Ovizio iLine F (Ovizio Imaging Systems NV/SA, Brussels, Belgium).

In certain embodiments, the monitoring is performed continuously for the duration of the incubation step. In some embodiments, the monitoring is performed in real time. In some embodiments, the monitoring is performed at discrete time points. In some embodiments, the monitoring is performed at least every 15 minutes for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 30 minutes for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 45 minutes for the duration of the incubation step. In some embodiments, the monitoring is performed at least every hour for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 2 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 4 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 6 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 8 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 10 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 12 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 14 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 16 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 18 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 20 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least every 22 hours for the duration of the incubation step. In some embodiments, the monitoring is performed at least once a day for the duration of the incubation step. In some embodiments, the monitoring is performed at least once every two days for the duration of the incubation step. In some embodiments, the monitoring is performed at least once for the duration of the incubation step.

In some embodiments, cellular characteristics that can be determined by monitoring, including using optical techniques such as DHM or DDHM, include cell viability, cell concentration, cell number, and/or cell density. In some embodiments, cell viability is characterized or determined. In some embodiments, cell concentration, density, and/or number are characterized or determined. In some embodiments, viable cell concentration, viable cell number, and/or viable cell density are characterized or determined.

In some embodiments, e.g., as the cells undergo incubation under culture conditions for expansion, the cells are monitored by an automated system until an expansion threshold is reached, e.g., 1, 2, 3, 4 or more population doublings. In some embodiments, once the expansion threshold is reached, the cells are harvested, e.g., by an automated or manual method, e.g., by a human operator. The expansion threshold can depend on the total concentration, density, and/or number of cultured cells as determined by the automated system. Alternatively, the expansion threshold can depend on the concentration, density, and/or number of viable cells.

In some embodiments, the harvested cells are formulated as described, e.g., in the presence of a pharma- ceutically acceptable carrier. In some embodiments, the harvested cells are formulated in the presence of a cryoprotectant.

F. Small Molecules in Process In some embodiments, provided herein is a method that includes manufacturing or producing engineered cells (e.g., CAR-T cells) in the presence of a regulator, thereby improving the persistence, lack of exhaustion and/or efficacy of the engineered cells produced or produced.In some aspects, the provided method produces a composition of cells, including primary T cells engineered to express a recombinant receptor, for use in, for example, cell therapy, that (i) contains fewer exhausted cells and/or fewer cells that exhibit exhaustion-related markers or phenotypes; (ii) contains an increased proportion of memory-like T cells, e.g., memory T cells with long life span; (iii) is less differentiated; (iv) shows improved or enhanced survival, expansion, persistence and/or anti-tumor activity; (v) shows improved therapeutic efficacy; and/or (vi) shows improved clinical response duration compared to the composition of engineered cells produced by an alternative method, e.g., an alternative method that is not performed in the presence of a regulator.In some embodiments, the comparison with the alternative process is made to the same process, except that it is not performed in the presence of a regulator.

In some embodiments, the modulating agent is contacted with the cell or cell population before the cells are harvested, harvested, or formulated (e.g., the modulating agent is in the cell or interacts with one or more cell surface molecules). In some embodiments, the modulating agent is present before, during, or after the cells are subjected to a stimulus, e.g., T cell activation. In some embodiments, the modulating agent is contacted with the cell or cell population before or during a stimulus, e.g., a stimulus described herein, such as in Sections I-C (e.g., the modulating agent is in the cell or interacts with one or more cell surface molecules). In some embodiments, the modulating agent is present before, during, or after the cells are subjected to a manipulation, e.g., transduction. In some embodiments, the modulating agent is contacted with the cell or cell population before or during a manipulation, e.g., a manipulation described herein, such as in Sections I-E (e.g., the modulating agent is in the cell or interacts with one or more cell surface molecules). In some embodiments, the modulating agent is contacted with the cell or cell population during or after incubation, e.g., incubation as described herein, such as in Section I-E-3 (e.g., the modulating agent is intracellular or interacts with one or more cell surface molecules). In some embodiments, the modulating agent is contacted with the cell or cell population during stimulation (e.g., stimulation as described herein, such as in Section I-C), manipulation (manipulation as described herein, such as in Section I-E), and/or incubation (e.g., incubation as described herein, such as in Section I-E-3) (e.g., the modulating agent is intracellular or interacts with one or more cell surface molecules). In certain embodiments, the cell or cell population is subjected to a process, procedure, step or technique in the presence of the modulating agent after incubation but before a step to harvest, harvest or formulate the cells. In certain embodiments, the cell or cell population is subjected to a process, procedure, step or technique in the presence of the modulating agent after incubation.

In some embodiments, the cells to be engineered (e.g., transduced) are contacted (e.g., incubated) with a modulating agent, e.g., in a culture medium, prior to engineering. In some embodiments, the cells are engineered in the presence of a modulating agent. In some embodiments, one or more engineered cells are contacted (e.g., incubated) with a modulating agent, e.g., in a culture medium, such as a basal medium that does not contain one or more recombinant cytokines or does not contain any recombinant cytokines. Also provided in some embodiments are compositions comprising (i) a modulating agent and (ii) cells to be engineered and/or cells subjected to engineering (including engineered cells), e.g., primary immune cells (e.g., T cells), during the manufacture or production of engineered cells, e.g., for cell-based therapies.

In some embodiments, the modulator is selected from the group consisting of PI3K inhibitors, Akt pathway, mTOR inhibitors, Ras/ERK inhibitors, NF-κΒ inhibitors, BET inhibitors, CDK inhibitors, CRAC channel inhibitors, Cox inhibitors, dopamine antagonists, ERK5 inhibitors, glucocorticoids, IGF-1R inhibitors, IKK inhibitors, JAK inhibitors, Lck inhibitors, PDKl inhibitors, Raf inhibitors, and Syk inhibitors. In some embodiments, the Src inhibitors include, but are not limited to, dasatinib, saracatinib, bosutinib, KX01, and revastinib (DCC-2036). In some embodiments, the Src inhibitors include revastinib (DCC-2036). Particular agents useful as modulators of the present disclosure are disclosed in WO2019018603, WO2018106595, and PCT/US2018/058812, which are incorporated by reference in their entireties herein.

In some embodiments, the modulator is or includes a compound, small molecule, e.g., a small organic molecule, polynucleotide, oligonucleotide, siRNA or polypeptide, or a fragment, isoform, variant, analog or derivative thereof, that inhibits, reduces, prevents and/or is capable of inhibiting, reducing or preventing one or more activity of a target, such as mTOR. In certain embodiments, the agent is a small molecule having a molecular weight of less than 10 kD, less than 9 kD, less than 8 kD, less than 7 kD, less than 6 kD, less than 5 kD, less than 4 kD, less than 3 kD, less than 2 kD, less than 1 kD, less than 0.5 kD or less than 0.1 kD.

In some embodiments, the modulator is or comprises an agent that inhibits mTOR activity. In some embodiments, the cells to be engineered (e.g., transduced) are contacted (e.g., incubated) with an mTOR inhibitor prior to engineering. In some embodiments, the cells are engineered in the presence of an mTOR inhibitor. In some embodiments, one or more engineered cells are contacted (e.g., incubated) with an mTOR inhibitor in a culture medium, such as a basal medium that does not contain one or more recombinant cytokines or does not contain any recombinant cytokines. Also provided in some embodiments are compositions comprising (i) an mTOR inhibitor and (ii) a cell to be engineered and/or a cell that has been subjected to engineering (including an engineered cell) during the manufacture or production of the engineered cell.

In some embodiments, an agent that inhibits mTOR activity can inhibit, reduce and/or decrease and/or inhibit, reduce and/or decrease at least one activity of mTOR. In certain embodiments, an agent that inhibits mTOR activity can inhibit, reduce and/or decrease and/or inhibit, reduce and/or decrease mTOR kinase activity. In some embodiments, an agent that inhibits mTOR activity can inhibit, reduce and/or decrease and/or inhibit, reduce and/or decrease mTORC1 activity, e.g., mTORC1 kinase activity and/or mTORC2 activity. In some embodiments, an agent that inhibits mTOR activity prevents the formation of an mTORC1 complex and/or destabilizes said complex. In certain embodiments, an agent that inhibits activity prevents the formation of an mTORC2 complex and/or destabilizes said complex.

In certain embodiments, the agent that inhibits mTOR activity inhibits the activity of at least one additional kinase. In certain embodiments, the at least one additional kinase is PI3K. In certain embodiments, the agent that inhibits mTOR activity (i) does not inhibit PI3K activity; (ii) does not detectably inhibit PI3K activity at the _IC50 of mTOR activity; and/or (iii) does not detectably inhibit PI3K at all concentrations that detectably inhibit mTOR activity. In some embodiments, the agent that inhibits mTOR activity inhibits, e.g., selectively inhibits, mTORC1 and mTORC2 kinase activity compared to PI3K activity. In certain embodiments, the agent that inhibits mTOR activity inhibits mTORC1 and mTORC2 kinase activity. In certain embodiments, the agent that inhibits mTOR activity selectively inhibits mTORC1 activity, e.g., mTORC1 kinase activity.

In certain embodiments, an agent that inhibits mTOR activity (i) does not inhibit mTORC2 activity; (ii) does not detectably inhibit mTORC2 activity at the _IC50 of mTORC1 activity; and/or (iii) does not detectably inhibit mTORC2 at all concentrations that detectably inhibit mTORC1 activity.

In some embodiments, the agent that inhibits mTOR activity is CC214-1 (Celgene), CC214-2 (Celgene), CC0470324, GDC0980, SAR245409, VS5584, PI-103, SF1126, BGT226, XL765, PF-04691502, dactolisib (codenamed NVP-BEZ235 and BEZ-235), pyrazolopyrimidine, torin ( l), Torkinib (PP242), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), DS3078a, Rapamycin (Sirolimus), Temsirolimus (CC1779), Everolimus (RAD001), Deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI). In some embodiments, the agent that inhibits mTOR activity has or includes a formula provided in Formula (I), Formula (II), or Formula (III). In some embodiments, the agent is Compound 155, Compound 246, or Compound 63.

［式中、R¹は、置換もしくは非置換のC_1-8アルキル、置換もしくは非置換のアリール、置換もしくは非置換のヘテロアリール、置換もしくは非置換のシクロアルキル、または置換もしくは非置換のヘテロシクロアルキルであり、R²は、置換もしくは非置換のC_1-8アルキル、置換もしくは非置換のアリール、置換もしくは非置換のヘテロアリール、置換もしくは非置換のシクロアルキル、または置換もしくは非置換のヘテロシクロアルキルであり、R³およびR⁴は、独立して、HまたはC_1-8アルキルである］に示される式を含む。いくつかの態様では、mTOR活性を阻害する作用物質は、式（I）の化合物またはその薬学的に許容される塩もしくは溶媒和物であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、式（I）の化合物またはその薬学的に許容される塩であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、2-(3-ヒドロキシフェニル)-9-(2-イソプロピルフェニル)-8-オキソ-8,9-ジヒドロ-7H-プリン-6-カルボキサミドまたはその薬学的に許容される塩もしくは溶媒和物であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、

またはその薬学的に許容される塩であるかそれを含む。 In certain embodiments, the agent has the formula (I):

[wherein R ¹ is substituted or unsubstituted C _1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; R ² is substituted or unsubstituted C _1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; R ³ and R ⁴ are independently H or C _1-8 alkyl]. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of formula (I) or a pharma- ceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of formula (I) or a pharma- ceutically acceptable salt thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises 2-(3-hydroxyphenyl)-9-(2-isopropylphenyl)-8-oxo-8,9-dihydro-7H-purine-6-carboxamide or a pharma- ceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is

or a pharma- ceutically acceptable salt thereof.

［式中、Lは、直接の結合、NHまたはOであり、Yは、NまたはCR³であり、式中、R¹は、H、置換もしくは非置換のC_1-8アルキル、置換もしくは非置換のC_2-8アルケニル、置換もしくは非置換のアリール、置換もしくは非置換のヘテロアリール、置換もしくは非置換のシクロアルキルまたは置換もしくは非置換のヘテロシクロアルキルであり、R²は、H、置換もしくは非置換のC_1-8アルキル、置換もしくは非置換のアリール、置換もしくは非置換のヘテロアリール、置換もしくは非置換のシクロアルキル、または置換もしくは非置換のヘテロシクロアルキルであり、R³は、H、置換もしくは非置換のC_1-8アルキル、置換もしくは非置換のアリール、置換もしくは非置換のヘテロアリール、置換もしくは非置換のシクロアルキル、置換もしくは非置換のヘテロシクロアルキル、-NHR⁴または-N(R⁴)₂であり、R⁴は、出現毎に、独立して、置換もしくは非置換のC_1-8 アルキル、置換もしくは非置換のアリール、置換もしくは非置換のヘテロアリール、置換もしくは非置換のシクロアルキル、または置換もしくは非置換のヘテロシクロアルキルである］に示される式を含む。いくつかの態様では、mTOR活性を阻害する作用物質は、式（II）の化合物またはその薬学的に許容される塩もしくは溶媒和物であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、6-(4-(2H-1,2,4-トリアゾール-3-イル)フェニル)-1-(2-(テトラヒドロ-2H-ピラン-4-イル)エチル)-1H-イミダゾ[4,5-b]ピラジン-2(3H)-オンまたはその薬学的に許容される塩もしくは溶媒和物であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、

またはその薬学的に許容される塩であるかそれを含む。 In some embodiments, the agent has the formula (II):

wherein L is a direct bond, NH or O; Y is N or ^CR3 ; wherein ^R1 is H, substituted or unsubstituted _C1-8 alkyl, substituted or unsubstituted _C2-8 alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl; ^R2 is H, substituted or unsubstituted _C1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl; ^R3 is H, substituted or unsubstituted _C1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, -NHR4 or -N( ^R4 )2; and ^R4 at each occurrence is independently substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, -NHR4 or -N( ^R4 ) ₂ ; In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of formula (II) or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises 6- ₍ 4-(2H-1,2,4-triazol-3-yl)phenyl)-1-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises

or a pharma- ceutically acceptable salt thereof.

［式中、R¹は、置換もしくは非置換のC_1-8アルキル、置換もしくは非置換のアリール、置換もしくは非置換のシクロアルキル、置換もしくは非置換のヘテロシクリル、または置換もしくは非置換のヘテロシクリルアルキルであり、R²は、H、置換もしくは非置換のC_1-8 アルキル、置換もしくは非置換のシクロアルキル、置換もしくは非置換のヘテロシクリル、置換もしくは非置換のヘテロシクリルアルキル、置換もしくは非置換のアラルキル、または置換もしくは非置換のシクロアルキルアルキルであり、R³は、H、または置換もしくは非置換のC_1-8 アルキルである］に示される式を含む。ある特定の態様では、R¹は、置換もしくは非置換のアリールまたは置換もしくは非置換のヘテロアリールである。いくつかの態様では、R¹は、置換されているピリジルである。いくつかの態様では、mTOR活性を阻害する作用物質は、式（III）の化合物またはその薬学的に許容される塩もしくは溶媒和物であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、式（III）の化合物またはその薬学的に許容される塩であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、7-(6-(2-ヒドロキシプロパン-2-イル)ピリジン-3-イル)-1-((1r,4r)-4-メトキシシクロヘキシル)-3,4-ジヒドロピラジノ[2,3-b]ピラジン-2(1H)-オンまたはその薬学的に許容される塩もしくは溶媒和物であるかそれを含む。いくつかの態様では、mTOR活性を阻害する作用物質は、

またはその薬学的に許容される塩であるかそれを含む。 In certain embodiments, the agent has the formula (III):

In the formula, ^R1 is substituted or unsubstituted _C1-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl; R2 is H, substituted or unsubstituted _C1-8 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkylalkyl; ^R3 is H, or substituted or unsubstituted _C1-8 alkyl. In certain embodiments, ^R1 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In some embodiments, ^R1 is substituted pyridyl. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of formula (III) or its ^pharma- ceutical acceptable salt or solvate. In some embodiments, the agent that inhibits mTOR activity is or comprises a compound of formula (III) or a pharma- ceutically acceptable salt thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1-((1r,4r)-4-methoxycyclohexyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one or a pharma- ceutically acceptable salt or solvate thereof. In some embodiments, the agent that inhibits mTOR activity is or comprises

or a pharma- ceutically acceptable salt thereof.

As will be appreciated by those skilled in the art, the scope of the present invention also includes analogs or derivatives of all other agents that are functionally classified into their respective classes based on their target, including, but not limited to, salts, esters, ethers, solvates, hydrates, stereoisomers, or prodrugs.

G. Cell Harvesting and Collection In some embodiments, cells are harvested or collected. In certain embodiments, cells are harvested or collected after completion of incubation as described in section IE-3. In certain embodiments, the harvested or collected cells are output population cells. In some embodiments, the output population comprises live CD3+, CD4+, CD8+ cells, and/or cells positive for recombinant receptors, e.g., CAR+. In certain embodiments, the harvested CD4+ T cells and the formulated CD8+ T cells are output CD4+ and CD8+ T cells. In certain embodiments, the formulated cell population, e.g., the formulated population of enriched CD4+ and CD8+ cells, is output cell population, e.g., the output population of enriched CD4+ and CD8+ cells.

In some embodiments, the cells or cell populations harvested, collected, or formulated have not undergone any expansion, e.g., any conditions in which the cells are incubated or cultured under conditions that increase the amount of viable cells during incubation or culture. For example, in some aspects, the harvested cells have not undergone any incubation or culture that increases the amount of total viable cells at the end of the incubation or culture compared to the number of total viable cells at the start of the incubation or culture. In some embodiments, the harvested cells have not undergone any incubation or culture step that is specifically intended to increase (e.g., expand) the total number of viable cells at the end of the incubation or culture process compared to the start of the incubation or culture process. In some embodiments, the cells are incubated or cultured under conditions that may result in expansion, but the incubation or culture conditions are not performed with the purpose of expanding the cell population. In some embodiments, the harvested cells may have undergone expansion despite being manufactured in a process that does not include an expansion step. In some embodiments, a manufacturing process that does not include an expansion step is referred to as a non-expansion process or a minimal expansion process. A "non-expansion" process may also be referred to as a "minimal expansion" process. In some embodiments, a non-expansion or minimal expansion process may result in cells that have undergone expansion despite the process not including a step for expansion. In some embodiments, the cells harvested may have undergone an incubation or culture step that includes a media composition designed to reduce, inhibit, minimize or eliminate expansion of the cell population as a whole. In some embodiments, the cells harvested, harvested or formulated have not previously undergone incubation or culture performed in a bioreactor or under conditions in which the cells are rocked, rotated, shaken or perfused for all or part of the incubation or culture.

In some embodiments, the cells or cell population harvested, collected, or formulated have undergone expansion, e.g., have been subjected to conditions in which the cells are incubated or cultured under conditions that increase the amount of viable cells during incubation or culture. For example, in some aspects, the harvested cells have undergone an incubation or culture in which the amount of total viable cells at the end of the incubation or culture is increased compared to the number of total viable cells at the beginning of the incubation or culture. In some embodiments, the harvested cells have undergone an incubation or culture step expressly for the purpose of increasing (e.g., expanding) the total number of viable cells at the end of the incubation or culture process compared to the beginning of the incubation or culture process. In some embodiments, the harvested cells have undergone an incubation or culture step that includes a media composition designed to encourage or promote the expansion of the entire cell population. In some embodiments, the cells to be harvested, harvested or formulated have previously undergone incubation or culture carried out in a bioreactor or under conditions in which the cells are rocked, rotated, shaken or perfused for all or a portion of the incubation or culture.

In some embodiments, the cell selection, isolation, separation, enrichment and/or purification steps are performed before the cells or cell population are harvested, collected or formulated. In some embodiments, the cell selection, isolation, separation, enrichment and/or purification steps are performed using chromatography as disclosed herein. In some embodiments, the chromatographic T cell selection step is performed after T cell transduction but before harvesting the cells, before collecting the cells and/or before formulating the cells. In some embodiments, the chromatographic T cell selection step is performed immediately prior to harvesting the cells.

In certain embodiments, the time from the start of stimulation (e.g., on-column stimulation) to the cells being harvested, harvested, or formulated is at or about 24 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or less than 24 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours. In some embodiments, the time from the start of stimulation to the cells being harvested, harvested or formulated to generate engineered cells is between or about 12 hours to 24 hours, 36 hours to 120 hours, 48 hours to 96 hours or 48 hours to 72 hours, inclusive. In particular embodiments, the time from the start of incubation to the cells being harvested, harvested or formulated is 48 hours, 72 hours or 96 hours, or about 48 hours, 72 hours or 96 hours, or less than 48 hours, 72 hours or 96 hours. In particular embodiments, the time from the start of incubation to the cells being harvested, harvested or formulated is 48 hours ± 6 hours, 72 hours ± 6 hours or 96 hours ± 6 hours. In certain embodiments, the time from the start of incubation to harvesting, collecting, or formulating the cells is at or about 96 hours or 4 days.

In certain embodiments, the cells are harvested, collected, or formulated in serum-free medium, such as those described in Section III of the present specification or in PCT/US2018/064627, which is incorporated herein by reference. In some embodiments, the cells are harvested, collected, or formulated in the same serum-free medium used during the incubation, e.g., as described in Section I-E-3 of the present specification.

In certain embodiments, the cells are harvested, collected, or formulated in a basal medium that does not contain one or more recombinant cytokines and does not contain serum components, i.e., serum-free medium, but may contain one or more additional components, such as those described in Section III.B. In certain embodiments, the use of such serum-free medium provides a nutrient-poor medium that is compatible with the maintenance of the cells but does not include certain factors that may activate the cells or render them metabolically active, thereby rendering the cells dormant or quiescent or likely to become dormant or quiescent. In some aspects, incubation in the presence of such serum-free medium allows for the recovery of the cells after stimulation (e.g., by Sections I-C) and genetic manipulation (e.g., transduction). In some aspects, harvesting, collecting, or formulating the cells in the presence of such serum-free medium results in the formulation of an output composition (e.g., a therapeutic cell composition) containing the cells that is less susceptible to damage or loss of viability, for example, when the harvested/formulated cells are cryopreserved and then thawed immediately prior to use. In some embodiments, the cells in the output composition (e.g., a therapeutic cell composition), when thawed, have lower levels of caspases or other markers of apoptosis than cells incubated in a similar medium but containing one or more recombinant cytokines, serum, or other factors that may render the cells metabolically more active upon cryopreservation of the output composition (e.g., a therapeutic cell composition).

In certain embodiments, one or more populations of enriched T cells are formulated. In particular embodiments, one or more populations of enriched T cells are formulated after the one or more populations have been manipulated and/or cultured. In particular embodiments, the one or more populations are input populations or output compositions (e.g., selected and stimulated cells). In some embodiments, one or more input populations or output compositions are previously cryoprotected and stored and are thawed prior to incubation (e.g., incubation as described in Section I-E-3).

In certain embodiments, the cells are harvested prior to 1, 2, 3, 4, 5, 6, 8, 10, 20 or more cell doublings, prior to about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 10, about 20 or more cell doublings of the cell population, or prior to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 20 or more cell doublings, e.g., doublings that occur during incubation.

In certain embodiments, the cells are harvested or collected at a time before the total number of cells, e.g., the total number of incubated cells or cells that have been incubated (e.g., incubated as described in Section I-E-3), exceeds 1, 2, 3, 4, 5, 6, 8, 10, 20, or more than 20 times the number of cells in the input population, e.g., the total number of cells contacted with a stimulatory reagent. In some embodiments, the cells are harvested or collected at a time before the total number of incubated cells exceeds 1, 2, 3, 4, 5, 6, 8, 10, 20, or more than 20 times the total number of transformed, transduced, or spinoculated cells, e.g., the total number of cells contacted with a viral vector. In certain embodiments, the cells are T cells, viable T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, T cells expressing a CAR, or any combination thereof. In certain embodiments, the cells are harvested or collected at a time before the total number of cells exceeds the total number of cells of the input population. In various embodiments, the cells are harvested or collected at a time before the total number of viable CD3+ T cells exceeds the total number of viable CD3+ cells of the input population. In certain embodiments, the cells are harvested or collected at a time before the total number of cells exceeds the total number of cells of the transformed, transduced, or spinoculated cells. In various embodiments, the cells are harvested or collected at a time before the total number of viable CD3+ T cells exceeds the total number of viable CD3+ cells of the transformed, transduced, or spinoculated cells.

In certain embodiments, the formulated cells are output cells. In some embodiments, the formulated population of enriched T cells is an output population of enriched T cells. In certain embodiments, the formulated CD4+ T cells and the formulated CD8+ T cells are output CD4+ and CD8+ T cells. In certain embodiments, the formulated cell population, e.g., the formulated population of enriched CD4+ and CD8+ cells, is an output cell population, e.g., an output population of enriched CD4+ and CD8+ cells.

In some embodiments, the cells can be formulated in a container, such as a bag or vial.

In some embodiments, the cells are formulated in a pharma- ceutically acceptable buffer, which may, in some aspects, include a pharma- ceutically acceptable carrier or excipient. In some embodiments, the treatment involves exchanging the medium for a pharma- ceutically acceptable or desired medium or formulation buffer for administration to a subject. In some embodiments, the treatment step may involve washing the transduced and/or expanded cells and replacing the cells in a pharma- ceutically acceptable buffer, which may include any one or more pharma- ceutically acceptable carriers or excipients. Examples of such pharmaceutical forms, including pharma- ceutically acceptable carriers or excipients, may be any of those described below in relation to forms acceptable for administering the cells and compositions to a subject. The pharmaceutical composition in some embodiments contains an amount of cells effective to treat or prevent a disease or condition, e.g., a therapeutically effective or prophylactically effective amount.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some aspects, the choice of carrier is determined in part by the particular cells and/or by the method of administration. Thus, a variety of suitable formulations exist. For example, the pharmaceutical composition may include a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; a sugar such as sucrose, mannitol, trehalose or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and/or a non-ionic surfactant such as polyethylene glycol (PEG).

In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

The formulation may include an aqueous solution. The formulation or composition may also contain two or more active ingredients useful for the particular indication, disease, or condition being treated with the cells, preferably those with complementary activities in the cells, provided that each activity does not adversely affect the other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.

In some embodiments, the compositions are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injectable solutions may be prepared by incorporating the cells in a suitable carrier, diluent, or excipient, e.g., a solvent, such as a mixture with sterile water, saline, glucose, dextrose, and the like. The compositions may contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, and/or coloring agents, depending on the desired route of administration and formulation. In some aspects, standard textbooks may be consulted to prepare suitable preparations.

Various additives can be added which enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

In some embodiments, the formulation buffer contains a cryopreservative. In some embodiments, the cells are formulated in a cryopreservation solution containing 1.0% to 30% DMSO solution, such as a 5% to 20% DMSO solution or a 5% to 10% DMSO solution. In some embodiments, the cryopreservation solution is or contains, for example, PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. In some embodiments, the cryopreservation solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the treatment step can involve washing the transduced and/or expanded cells and replacing them with cells in a cryopreservative solution. In some embodiments, the cells are frozen, e.g., cryoprotected or cryopreserved, in medium and/or solutions having a final concentration of 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5% or 5.0% DMSO or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5% or 5.0% DMSO, or between 1% and 15%, 6% and 12%, 5% and 10%, or 6% and 8% DMSO. In certain embodiments, the cells are frozen, e.g., cryoprotected or cryopreserved, in medium and/or solutions having a final concentration of, e.g., 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25% HSA or about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5% or 0.25% HSA, or 0.1%-5%, 0.25%-4%, 0.5%-2%, or 1%-2% HSA.

In certain embodiments, compositions of enriched T cells, e.g., stimulated, engineered, and/or cultured T cells, are formulated, cryoprotected, and then stored for a certain amount of time. In certain embodiments, the formulated cryoprotected cells are stored until the cells are released for infusion. In certain embodiments, the formulated cryoprotected cells are stored for 1 day to 6 months, 1 month to 3 months, 1 day to 14 days, 1 day to 7 days, 3 days to 6 days, 6 months to 12 months, or longer than 12 months. In some embodiments, the cells are cryoprotected and stored for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or for less than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In certain embodiments, after storage, the cells are thawed and administered to a subject. In certain embodiments, the cells are stored for 5 days or about 5 days. In some embodiments, the formulated cells are not cryoprotected.

In some embodiments, formulation is performed using one or more processing steps including washing, diluting or concentrating the cells, such as cultured or expanded cells. In some embodiments, processing can include diluting or concentrating the cells to a desired concentration or number, such as a unit dose form composition that includes the number of cells for administration at a given dose or a fraction thereof. In some embodiments, the processing step includes volume reduction, thereby allowing the concentration of the cells to be increased, if desired. In some embodiments, the processing step includes volume addition, thereby allowing the concentration of the cells to be decreased, if desired. In some embodiments, processing includes adding an amount of formulation buffer to the transduced and/or expanded cells. In some embodiments, the volume of the formulation buffer is at least 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL, or at least about 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL, or about 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL, or about 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL, or about 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL, mL to 1000 mL or approximately 10 mL to 1000 mL.

In some embodiments, such processing steps for formulating the cell composition are performed in a closed system. Examples of such processing steps may be performed using a centrifuge chamber in combination with one or more systems or kits related to a cell processing system, such as the centrifuge chambers manufactured and sold by Biosafe SA, including for use with the Sepax® or Sepax 2® cell processing systems. Exemplary systems and methods are described in International Publication WO2016/073602. In some embodiments, the method includes removing a formulated composition, which is a product composition of cells formulated in a formulation buffer, such as a pharma- ceutically acceptable buffer, from the internal cavity of the centrifuge chamber in any of the above-mentioned embodiments. In some embodiments, the removal of the formulated composition is to a container, such as a bag, that is operably coupled to the centrifuge chamber as part of the closed system. In some embodiments, the container, such as a bag, is connected to the system at an output line or output location.

In some embodiments, a closed system, such as one associated with a centrifuge chamber or cell processing system, includes a multi-port output kit including a multi-way tubing manifold connected with a port at each end of a tubing line, to which one or more containers can be connected for removal of the formulated composition. In some aspects, a desired number or number of output containers, e.g., bags, can be sterilely connected to one or more, typically two or more, ports of a multi-port output, such as at least three, four, five, six, seven, eight or more. For example, in some embodiments, one or more containers, e.g., bags, can be attached to a port, or to fewer than all ports. Thus, in some embodiments, the system can provide for removal of the output composition into multiple output bags.

In some aspects, cells can be removed into one or more of a plurality of output bags in dosage administration amounts, such as single dose administration or multiple dose administration. For example, in some embodiments, the output bags can each contain a number of cells for administration at a given dose or fraction thereof. Thus, in some aspects, each bag may contain a single dose for administration or may contain a fraction of a desired dose such that two or more of the plurality of output bags, such as two output bags, or three output bags, collectively constitute one dose for administration.

Thus, the container, e.g., output bag, generally contains the cells to be administered, e.g., one or more unit doses thereof. A unit dose can be the amount or number of cells to be administered to a subject, or twice (or more) the number of cells to be administered. It can be the lowest dose or lowest possible dose of cells to be administered to a subject.

In some embodiments, each of the containers, e.g., bags, contains a separate unit dose of cells. Thus, in some embodiments, each container contains the same, or about the same, or substantially the same number of cells. In some embodiments, each unit dose contains at least or at least about 1×10 ⁶ , 2×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 5×10 ⁷ , or 1×10 ⁸ engineered cells, whole cells, T cells, or PBMCs. In some embodiments, the volume of the formulated cell composition in each bag is between 10 mL and 100 mL, e.g., at least or at least about 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL.

In some embodiments, such cells produced by this method, or compositions comprising such cells, are administered to a subject to treat a disease or condition.

H. Removal of Stimulating Reagents In some embodiments, the stimulating reagent (e.g., oligomeric stimulating reagent) is removed or separated from the collected cells or cell population after the cells are harvested, harvested, or formulated. In some embodiments, the stimulating reagent is removed or separated from the cells or cell population after collection from the chromatography column, such as after the elution step and cell harvesting step described in Section ID. In some embodiments, the stimulating reagent is removed or separated from the cells or cell population after or during incubation, such as the incubation described herein in Section IE-3. In certain embodiments, the cells or cell population undergoes a process, procedure, step, or technique after incubation but prior to the cell harvesting, harvesting, or formulation step to remove the stimulating reagent (e.g., oligomeric stimulating reagent). In certain embodiments, the cells or cell population undergoes a process, procedure, step, or technique after incubation to remove the stimulating reagent (e.g., oligomeric stimulating reagent). In some aspects, if the stimulating reagent (e.g., oligomeric stimulating reagent) is separated or removed from the cells during incubation, the cells are returned to the same incubation conditions prior to separation or removal for the remainder of the incubation period.

In certain embodiments, the stimulating reagent (e.g., oligomeric stimulating reagent) is removed and/or separated from the cells. Without wishing to be bound by any theory, certain embodiments contemplate that binding and/or association between the stimulating reagent (e.g., oligomeric stimulating reagent) and the cells may, in some circumstances, be reduced over time during incubation. In certain embodiments, one or more agents may be added to reduce binding and/or association between the stimulating reagent and the cells. In certain embodiments, binding and/or association between the stimulating reagent and the cells may be reduced by, for example, a change in cell culture conditions, such as the addition of an agent (e.g., a competitor or a free binder). Thus, in some embodiments, the stimulating reagent (e.g., oligomeric stimulating reagent) may be removed from the incubation, cell culture system, and/or solution separately from the cells, for example, without removing the cells from the incubation, cell culture system, and/or solution.

In certain embodiments, the stimulating reagent (e.g., oligomeric stimulating reagent) is separated and/or removed from the cells after a period of time. In particular embodiments, the period of time is a period of time from the start of stimulation. In particular embodiments, the start of incubation is considered to be at or about the time when the cells are contacted with the stimulating reagent and/or the medium or solution containing the stimulating reagent. In particular embodiments, the stimulating reagent is removed or separated from the cells within 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours (inclusive) of the start of stimulation. In particular embodiments, the stimulating reagent (e.g., oligomeric stimulating reagent) is removed or separated from the cells at or about 48 hours after stimulation is initiated. In certain embodiments, the stimulating reagent is removed or separated from the cells at or about 72 hours after stimulation is initiated. In some embodiments, the stimulating reagent is removed or separated from the cells at or about 96 hours after stimulation is initiated.

1. Removal of Oligomeric Stimulatory Reagents In some embodiments, a population of stimulator cells generated or produced according to any of the methods provided herein (i.e., cells that have been subjected to column chromatography and on-column stimulation selection as described herein) is subjected to the addition of a substance, such as a competitor or free binder, to reduce and/or terminate signaling of one or more stimulatory substances. In some embodiments, the addition of the competitor or free binder is performed after the elution step as described herein (see Section ID). In some embodiments, the addition of the competitor or free binder is performed after the genetic engineering step as described herein. In some embodiments, the addition of the competitor or free binder is performed after the harvesting step as described herein. Thus, in some embodiments, the population of stimulated cells contains the presence of a substance, such as a competitor, such as biotin or a biotin analog, such as D-biotin. In some embodiments, the substance, e.g., a competitor such as biotin or a biotin analog, e.g., D-biotin, is present in an amount at least 1.5-fold greater, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 1000-fold, or more greater than the amount of the substance in a reference population or preparation of cultured cells (e.g., T cells) to which the substance was not exogenously added during one of the above-mentioned steps. In some embodiments, the amount of the substance, e.g., a competitor such as biotin or a biotin analog, e.g., D-biotin, in the population of stimulator cells is between 10 μM and 100 μM, 100 μM and 1 mM, 100 μM and 500 μM, or 10 μM and 100 μM, or about 10 μM and 100 μM, 100 μM and 1 mM, 100 μM and 500 μM, or 10 μM and 100 μM. In some embodiments, 10 μM or about 10 μM biotin or a biotin analog, such as D-biotin, is added to the cell or cell population to separate or remove the oligomeric stimulating reagent from the cell or cell population. In some embodiments, 1 mM or about 1 mM biotin or a biotin analog, such as D-biotin, is added to the cell or cell population to separate or remove the oligomeric stimulating reagent from the cell or cell population. In some embodiments, 1 mM or about 1 mM D-biotin is added to the cell or cell population to separate or remove the oligomeric stimulating reagent from the cell or cell population.

In certain embodiments, one or more stimulatory agents (e.g., agents that stimulate or activate TCRs and/or costimulatory molecules) are reversibly bound to, or otherwise associated with, the oligomeric reagent, such as through multiple specific binding sites (e.g., binding site Z) present on the oligomeric reagent. In some cases, this allows the stimulatory agents to be spaced closely together such that an avidity effect can occur when a target cell bearing (at least two copies of) a cell surface molecule that is bound or recognized by the stimulatory agent is contacted with the agent. In some aspects, the stimulatory agent has low affinity for the cell's molecule at binding site B, such that the receptor-binding reagent dissociates from the cell in the presence of a competing reagent. Thus, in some embodiments, the stimulatory agent is removed from the cell in the presence of a competing reagent.

In some embodiments, the oligomeric stimulating reagent is a streptavidin mutein oligomer having a reversibly attached anti-CD3 Fab and an anti-CD28 Fab. In some embodiments, the attached Fab contains a streptavidin binding domain that can be reversibly attached to the streptavidin mutein oligomer, for example. The anti-CD3 Fab and the anti-CD28 Fab are closely spaced such that an avidity effect can occur when a T cell expressing CD3 and/or CD28 is brought into contact with the oligomeric stimulating reagent having the reversibly attached Fab. In some aspects, the Fab has low affinity for CD3 and CD28, such that the Fab is separated from the cell in the presence of a competing reagent, for example, biotin or a biotin variant or biotin analog. Thus, in some embodiments, the Fab is removed or separated from the cell in the presence of a competing reagent, for example, D-biotin.

In some embodiments, the oligomer stimulating reagent, e.g., the oligomer stimulating streptavidin mutein reagent, is removed or separated from the cell or cell population prior to harvesting or formulating the cells. In some embodiments, the oligomer stimulating reagent, e.g., the oligomer stimulating streptavidin mutein reagent, is removed or separated from the cell or cell population after or during incubation, e.g., the incubations described herein, e.g., in Section I-F or Section I-E-3, by contacting or exposing the cell or cell population to a competing reagent, e.g., biotin or a biotin analogue, e.g., D-biotin. In certain embodiments, the cell or cell population is contacted or exposed to a competing reagent, e.g., biotin or a biotin analogue, e.g., D-biotin, and the oligomer stimulating reagent, e.g., the stimulating oligomer streptavidin mutein reagent, is removed after incubation but prior to genetically manipulating, harvesting, or formulating the cells. In certain embodiments, the cells or cell populations are contacted or exposed to a competing reagent, e.g., biotin or a biotin analog, e.g., D-biotin, and after incubation, the oligomer stimulating reagent, e.g., oligomer stimulating streptavidin mutein reagent, is removed. In some aspects, if the oligomer stimulating reagent, e.g., oligomer stimulating streptavidin mutein reagent, is separated or removed from the cells during incubation, e.g., by contacting or exposing it to a competing reagent, e.g., biotin or a biotin analog, e.g., D-biotin (see Section I-E-3), the cells are returned to the same incubation conditions prior to separation or removal for the remainder of the incubation period.

In some embodiments, to remove or separate the oligomeric stimulatory reagent from the cells, the cells are incubated with 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM of competing reagent and approximately 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, , 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM of competing reagent, or with at least 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM of competing reagent. In various embodiments, the reversibly attached anti-CD3 Fab and anti-CD28 To remove or separate the Fab-bearing stimulatory streptavidin mutein oligomers from the cells, the cells are incubated with 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM biotin or a biotin analogue such as D-biotin, or with approximately ... In one embodiment, the antibody is contacted with at least 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM biotin or a biotin analogue such as D-biotin, or with at least 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 100 μM, 500 μM, 0.01 μM, 1 mM, or 10 mM biotin or a biotin analogue such as D-biotin. In various embodiments, to remove or separate a stimulatory oligomeric reagent, such as a streptavidin mutein oligomer having reversibly attached anti-CD3 Fab and anti-CD28 Fab, from the cells, the cells are contacted with, for example, 100 μM to 10 mM, or about 100 μM to 10 mM, such as 1 mM, biotin or a biotin analog, such as D-biotin. In various embodiments, the cells are contacted with, for example, 100 μM to 10 mM, or about 100 μM to 10 mM, such as 1 mM, biotin or a biotin analog, such as D-biotin, for 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours after contact or exposure to D-biotin.

In certain embodiments, the oligomer stimulating reagent, e.g., the oligomer stimulating streptavidin mutein reagent, is removed or separated from the cells within 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, or 12 hours (inclusive) of the initiation of stimulation. In certain embodiments, the oligomer stimulating reagent, e.g., the oligomer stimulating streptavidin mutein reagent, is removed or separated from the cells at or about 48 hours after stimulation is initiated. In certain embodiments, the oligomer stimulating reagent, e.g., the oligomer stimulating streptavidin mutein reagent, is removed or separated from the cells at or about 72 hours after stimulation is initiated. In some embodiments, the oligomer stimulating reagent, such as the oligomer stimulating streptavidin mutein reagent, is removed or separated from the cells at or about 96 hours after stimulation is initiated.

In certain embodiments, the cells or cell populations are contacted or exposed to a competing reagent, e.g., biotin or a biotin analog, e.g., D-biotin, and the stimulatory oligomer reagent, e.g., a stimulatory oligomer streptavidin mutein reagent, is removed during or after the incubation described herein, e.g., in Section I-E-3, at or about 48 hours or at or about 2 days after stimulation begins. In some aspects, when the stimulatory oligomer reagent, e.g., a stimulatory oligomer streptavidin mutein reagent, is separated or removed from the cells during the incubation, e.g., by contacting or exposing it to a competing reagent, e.g., biotin or a biotin analog, e.g., D-biotin, the cells are returned to the same incubation conditions as before separation or removal, for the remainder of the incubation. In another aspect, when the stimulatory oligomer reagent, e.g., stimulatory oligomer streptavidin mutein reagent, is separated or removed from the cells after incubation, e.g., by contacting or exposing to a competing reagent, e.g., biotin or a biotin analogue, e.g., D-biotin, the cells are further incubated for or about 2, 6, 12, 18, 24, 30, 36, 42, or 48 hours after contacting or exposing to the competing reagent. In some embodiments, the transduced cells with D-biotin treatment are further incubated for or about 24±6 hours after D-biotin addition. In some embodiments, the transduced cells with D-biotin treatment are further incubated for or about 48 hours after D-biotin addition.

I. Sequential Selection, Parallel Selection, and Polishing The methods provided herein allow for multiple selection steps, e.g., by column chromatography, to isolate and/or enrich a target cell population (e.g., T cells, CD3+, CD4+, CD8+ T cells). In some embodiments, one or more selection steps are performed at one or more points or after certain steps of the process to create an output composition of engineered cells (e.g., a therapeutic cell composition), e.g., as described in sections IA-H above. In some embodiments, a selection step performed after an initial cell selection, e.g., as described in sections IB and IC, is referred to as a polishing step. Polishing steps may be performed for a variety of purposes, including, but not limited to, to further purify the cell composition, select for specific cell subtypes (e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells), remove dead cells (e.g., select for viable cells), select for successfully engineered cells (e.g., cells expressing a transgene (e.g., a chimeric antigen receptor (CAR), T cell receptor (TCR), etc.), or adjust the ratio, total number, or concentration of specific cell types (e.g., CD8+ cells versus CD4+, CAR+ or TCR+ cells versus CAR- or TCR- cells, or total numbers or concentrations of CD4+, CD8+, CAR+, TCR+, and/or viable cells). In some embodiments, selection steps (e.g., polishing steps) are useful to increase product control and/or reduce inter-patient variability.

In some embodiments, the selection step (e.g., initial selection step and/or polishing step) includes multiple selection steps, e.g., to further purify the cell composition, select specific cell subtypes, select viable cells, select engineered cells, and/or adjust the ratio, total number, or concentration of cells. In some embodiments, the selection step (e.g., polishing step) is performed prior to incubation, e.g., incubation as described in Sections I-E-3 and/or I-F. In some embodiments, the selection step (e.g., polishing step) is performed prior to collection and recovery, e.g., prior to collection and recovery as described in I-H.

In some aspects, such methods (e.g., selection steps (e.g., initial selection and/or polishing steps)) are accomplished in a single process stream, such as in a closed system, by using sequential selections as provided herein, in which multiple different cell populations (e.g., output composition of stimulated and/or engineered cells) from a sample are enriched and/or isolated. In some aspects, performing separation or isolation in the same container or set of containers, e.g., set of tubes, is accomplished by performing successive positive and negative selection steps, subsequent steps subjecting the negative and/or positive fractions from the preceding steps to further selection, where the entire process is performed in the same tube or set of tubes. In one embodiment, a sample containing target cells (e.g., output composition of stimulated and/or engineered cells) is subjected to sequential selections, performing a first selection to enrich for one of the CD4+ or CD8+ populations, and using unselected cells from the first selection as a cell source for the second selection to enrich for the other of the CD4+ or CD8+ populations. In some embodiments, one or more further selections can be performed to enrich for one or both subpopulations of the CD4+ or CD8+ population, e.g., central memory T (T _CM ) cells or naive T cells. In some embodiments, specific subpopulations of T cells (e.g., CD3+, CD4+, CD8+ cells), such as cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are selected by positive or negative selection techniques during the selection step (e.g., initial selection step and/or polishing step). In some embodiments, the cell population containing target cells (e.g., the output composition of stimulated and/or engineered cells) is subjected to continuous selection, where a polishing step selects for viable cells. In some embodiments, the polishing step allows the ratio or total number of cells in the cell composition to be controlled or adjusted.

In one embodiment, a sample containing target cells (e.g., an output composition of stimulated and/or engineered cells) is subjected to sequential selections where a first selection is performed to enrich for a CD3+ population. In some embodiments, a further selection or selections can be performed to enrich for a subpopulation of the CD3+ population, e.g., CD4+ cells. In some embodiments, a further selection or selections can be performed to enrich for a subpopulation of the CD3+ population, e.g., CD8+ cells. In some embodiments, a further selection or selections can be performed to enrich for viable cells. In some embodiments, a further selection or selections can be performed to enrich for a subpopulation of CD3+ cells, e.g., viable CD3+CD4+ and/or CD3+CD8+ cells. In some embodiments, selecting for viable cells comprises or consists of removing dead cells from the cell population (e.g., an output composition of stimulated and/or engineered cells or subpopulations thereof).

In some embodiments, the methods disclosed herein (e.g., the selection steps (e.g., initial selection and/or polishing steps)) need not be performed using sequential selection techniques. In some embodiments, the methods disclosed herein (e.g., the selection steps (e.g., initial selection and/or polishing steps)) can be performed using sequential selection techniques combined with parallel selection techniques. In some embodiments, the selection steps (e.g., initial selection and/or polishing steps) may not use sequential selection or may use sequential selection that is not performed in a closed system or in a set of vessels using the same tubing. In some embodiments, the selection steps (e.g., initial selection and/or polishing steps) are accomplished in a single step, e.g., using a single chromatography column. In some embodiments, the selection steps (e.g., initial selection and/or polishing steps) are accomplished using parallel selection techniques. For example, the selection steps (e.g., initial selection and/or polishing steps) are accomplished by simultaneously performing positive and/or negative selection steps, e.g., in a closed system where the entire process is performed in the same tube or set of tubes. In some embodiments, a sample containing target cells (e.g., an output composition of stimulated and/or engineered cells) is subjected to parallel selection in which the sample (e.g., an output composition of stimulated and/or engineered cells) is loaded onto two or more chromatography columns, where each column performs a selection of a cell population. In some embodiments, the two or more chromatography columns perform selection of CD3+, CD4+, or CD8+ populations individually. In some embodiments, the two or more chromatography columns perform selection of the same cell population. For example, the two or more chromatography columns may perform selection of CD3+ cells. In some embodiments, the two or more chromatography columns, including affinity chromatography or gel permeation chromatography, perform selection of the same cell population independently. In some embodiments, the two or more chromatography columns, including affinity chromatography or gel permeation chromatography, perform selection of different cell populations independently. In some embodiments, a further selection or selections can be performed to enrich for subpopulations of one or all of the cell populations selected via parallel selection. For example, the selected cells may be further selected for central memory T (T _CM ) cells or naive T cells. In some embodiments, a sample (e.g., an output composition of stimulated and/or engineered cells) containing target cells (e.g., CD3+ cells) is subjected to parallel selection, where parallel selection is performed to enrich for CD4+ and CD8+ populations. In some embodiments, one or more additional selections can be performed to enrich for subpopulations of the CD4+ and CD8+ populations, e.g., central memory T (T _CM ) cells or naive T cells. In some aspects, it is contemplated that specific subpopulations of T cells (e.g., CD3+, CD4+, CD8+ T cells), such as cells positive for or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are selected by positive or negative selection techniques. In some embodiments, a sample (e.g., an output composition of stimulated and/or engineered cells) containing target cells (e.g., CD3+ cells) is subjected to parallel selection, where the parallel selection is performed to enrich for central memory T (T _CM ) cells or naive T cells. In some embodiments, a further selection or selections can be performed to enrich for subpopulations of central memory T (T _CM ) cells or naive T cells, e.g., CD4+, CD3+, or CD8+ cells. In some embodiments, the further selection is performed via sequential selection techniques after the parallel selection is accomplished.

In some embodiments, the selection step (e.g., initial selection and/or polishing step) can be performed using beads labeled with a selection agent described herein, and the positive and negative fractions from the first selection step can be retained followed by further positive selection of the positive fractions to enrich for the second selection marker by using beads labeled with a second selection agent or by subjecting the positive fractions to column chromatography as described above. In some embodiments, one or more polishing steps are performed using column chromatography as described herein, such as chromatography as described in Section I-B and/or chromatography including agent and reagent systems as described in Sections I-B and II. In some embodiments, the selection step (e.g., initial selection and/or polishing step) is accomplished using one or more methods including bead separation and column chromatography. In some embodiments, the selection step (e.g., initial selection and/or polishing step) is accomplished using column chromatography.

In some aspects, isolating multiple populations in a single or the same isolation or separation vessel or vessel set, e.g., a single column or set of columns, and/or the same tube or set of tubes, or using the same separation matrix or medium or reagent, e.g., the same magnetic matrix, affinity-labeled solid support, or antibody or other binding partner, includes features that streamline the isolation, e.g., resulting in reduced cost, time, complexity, sample handling requirements, resource, reagent, or equipment use. In some aspects, such features are advantageous in that they minimize the cost, efficiency, time, and/or complexity associated with the method and/or avoid potential harm to the cell product, e.g., harm caused by infection, contamination, and/or temperature change. The methods provided herein allow for multiple selection steps to enrich target populations both before or after cell selection in combination with on-column stimulation.

The methods provided herein further allow for the selection and enrichment of successfully stimulated and engineered cells. For example, in some embodiments, the serial, parallel, or single selection procedures described above may be used to identify stimulated cells expressing a recombinant receptor (e.g., CAR, TCR). In some embodiments, successfully engineered cells can be selected by using a selection agent capable of specifically binding to a surrogate marker (see, e.g., Section IV-A-1). In some embodiments, cells expressing a recombinant receptor (e.g., CAR) can be further enriched (e.g., polished) for subpopulation cells, e.g., CD4+ CAR+ T cells, CD8+ CAR+ T cells, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD45RA+, CD45RO+ T cells, and/or viable cells. In some embodiments, the selection process (e.g., initial selection and/or polishing process) allows for control or adjustment of the ratio, concentration, or total number of cells expressing the recombinant receptor (e.g., CAR, TCR) and/or subpopulations thereof. In some embodiments, the enriched (e.g., polished) population can be formulated for use (e.g., administration) in cell therapy.

J. Exemplary Features of the Process and/or Output Population In certain embodiments, the methods provided are used in connection with a process for producing or generating an output population of engineered T cells (e.g., a therapeutic cell population) from one or more input populations, e.g., from an input population obtained, selected, or enriched from a single biological sample. In certain embodiments, the output population contains cells expressing a recombinant receptor, e.g., a TCR or CAR. In certain embodiments, the cells of the output population are suitable for administration to a subject as a therapy, e.g., an autologous cell therapy.

In certain embodiments, the provided methods are used in conjunction with an entire process for thereby generating or producing output cells and/or output populations of engineered T cells, such as a process that includes some or all of the following steps: selecting and stimulating cells in a single step using column chromatography; collecting cells that have spontaneously detached without the use of a competitive reagent; engineering, transforming, transducing or transfecting the stimulator cells to express or contain a heterologous or recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor such as a CAR; incubating the cells, removing or separating the stimulator reagent (e.g., an oligomeric stimulator reagent) from the cells, harvesting and collecting the cells, and in some aspects thereby generating an output population of engineered T cells.

In some embodiments, the provided methods are used in conjunction with an entire process for generating or producing output cells and/or an output composition of enriched T cells, such as a process that includes some or all of the following steps: collecting or obtaining a biological sample; isolating, selecting, or enriching input cells from the biological sample; cryo-freezing and storing, then thawing the input cells; selecting and stimulating the cells using column chromatography in a single step; collecting cells that have spontaneously detached without the use of competitive reagents; genetically engineering the stimulator cells to express or contain a heterologous or recombinant polynucleotide, such as a polynucleotide encoding a recombinant receptor, such as a CAR; formulating the cultured cells into an output composition; and cryo-freezing and storing the formulated output cells until the cells are released for infusion and/or administration to a subject. In some embodiments, the provided methods do not include a step of expanding or increasing the number of cells during the process, such as by culturing the cells in a bioreactor under conditions that allow the cells to expand to a threshold amount, which is at least 3-fold, 4-fold, 5-fold, or more of the amount, level, or concentration of cells compared to the input population. In some embodiments, the provided methods include expanding or increasing the number of cells in the process by, for example, incubating or culturing the cells in a bioreactor under conditions that expand the cells to a threshold amount, which is at least 2-fold, 3-fold, 4-fold, 5-fold, or more, amount, level, or concentration of cells compared to the input population. In some embodiments, genetically engineering the cells is or includes a step to transduce the cells with a viral vector, for example, by spinoculating the cells in the presence of viral particles and then incubating the cells under static conditions in the presence of viral particles.

In certain embodiments, the duration of the entire process provided for the production of engineered cells from the start of stimulation to collection, harvesting, or formulation of the cells is, is about, or is less than 36, 42, 48, 54, 60, 72, 84, 96, 108, or 120 hours. In some embodiments, the duration of the entire process provided for the generation of engineered cells comprises, or is about, 36 to 120 hours, 48 to 96 hours, or 48 to 72 hours, inclusive, from the start of stimulation to harvesting, harvesting, or formulation of the cells. In particular embodiments, the fixed time to complete the provided process, measured from the start of incubation to harvesting, harvesting, or formulation of the cells, is, is about, or is less than 48, 72, or 96 hours. In particular embodiments, the fixed time to complete the provided process, measured from the start of incubation to harvesting, harvesting, or formulation of the cells, is 48 hours ±6 hours, 72 hours ±6 hours, or 96 hours ±6 hours.

In some embodiments, the incubation is completed at or about 24 hours to 120 hours, 36 hours to 108 hours, 48 hours to 96 hours, or 48 hours to 72 hours (inclusive) after the start of stimulation. In some embodiments, the incubation is completed at or about 120 hours, 108 hours, 96 hours, 72 hours, 48 hours, or 36 hours after the start of stimulation, or within 120 hours, 108 hours, 96 hours, 72 hours, 48 hours, or 36 hours. In certain embodiments, the incubation is completed after 24 hours ± 6 hours, 48 hours ± 6 hours, or 72 hours ± 6 hours.

In some embodiments, the entire process is performed with a single population of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and CD8+ T cells. In certain embodiments, the process is performed with two or more input populations of enriched T cells that are combined prior to and/or during the process to generate or create a single output population of enriched T cells. In some embodiments, the enriched T cells are or include engineered T cells, e.g., T cells transduced to express a recombinant receptor.

In some embodiments, an output population, e.g., a population of engineered T cells, is generated by (i) incubating a sample of T cells or a sample containing T cells under stimulatory conditions (e.g., on-column stimulation) on a chromatography column for less than 24 hours, (ii) introducing a heterologous or recombinant polynucleotide encoding a recombinant receptor into the stimulated population of T cells, (iii) incubating the cells, and then (iv) harvesting or harvesting the incubated cells.

In certain embodiments, an output population, e.g., a population of engineered T cells, is generated by (i) selecting and stimulating T cells in a single step using column chromatography (e.g., on-column stimulation) and collecting cells that spontaneously detach without the use of competitive reagents in less than 24 hours, (ii) transducing stimulated T cells with a viral vector encoding a recombinant receptor, e.g., by spinoculating stimulated T cells in the presence of the viral vector, (iii) incubating the transduced T cells under static conditions for or from 18 hours to 96 hours, inclusive, and (iv) harvesting the transformed population of T cells within or about 36-108 hours after incubation under stimulatory conditions begins.

In certain embodiments, an output population, e.g., a population of engineered T cells, is generated by (i) selecting and stimulating T cells in a single step using column chromatography (e.g., on-column stimulation) and collecting cells that spontaneously detach without the use of competitive reagents in less than or about 6 hours, (ii) transducing the stimulated T cells with a viral vector encoding a recombinant receptor for 1 hour or about 1 hour, (iii) incubating the transduced T cells for 72 hours or about 72 hours, and (iv) harvesting the transduced population of T cells within or about 90±10 hours after incubation under stimulatory conditions begins.

In some embodiments, the processes associated with the provided methods are compared to alternative processes. For example, in some embodiments, the methods provided herein are compared to alternative processes that contain steps for expanding cells. In some embodiments, the alternative process is a process that includes separate steps for cell selection and cell stimulation. In certain embodiments, the alternative process may differ in one or more specific aspects, but otherwise contain similar or similar features, aspects, steps, stages, reagents and/or conditions of the processes associated with the provided methods. In some embodiments, the alternative process is similar to the process associated with the provided methods, e.g., lacking or not including expansion culture, but differs in a manner including, but not limited to, one or more of the following: separate steps for selection and stimulation, including different reagents and/or media formulations; the presence of serum during incubation, transduction, transfection, and/or culture; a different cellular composition of the input population, e.g., the ratio of CD4+ T cells to CD8+ T cells; different stimulation conditions and/or different stimulating reagents; a different ratio of stimulating reagent to cells; a different vector and/or a different method of transduction; a different timing and order for incubating, transducing, and/or transfecting the cells; the absence or difference in one or more recombinant cytokines present during incubation or transduction (e.g., different cytokines or different concentrations), or a different timing for harvesting or collecting the cells.

In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, enrichment, and/or selection of input cells (e.g., CD4+ T cells or CD8+ T cells) from a biological sample to the time the output cells are collected, formulated, and/or cryoprotected, is less than 48 hours, 72 hours, 96 hours, 120 hours, 4 days, 5 days, 7 days, or 10 days, about 48 hours, 72 hours, 96 hours, 120 hours, 4 days, 5 days, 7 days, or 10 days, or less than 48 hours, 72 hours, 96 hours, 120 hours, 4 days, 5 days, 7 days, or 10 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, enrichment, and/or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the output cells are collected, formulated, and/or cryoprotected, is 4-5 days, about 4-5 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, enrichment, and/or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the output cells are collected, formulated, and/or cryoprotected, is 5 days or about 5 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, enrichment, and/or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the output cells are collected, formulated, and/or cryoprotected, is less than 5 days. In some embodiments, the duration or amount of time required to complete the provided process, measured from the isolation, enrichment, and/or selection of input cells (e.g., CD4+ or CD8+ T cells) from a biological sample to the time the output cells are collected, formulated, and/or cryoprotected, is 4 days or about 4 days. In some embodiments, the isolated, selected or enriched cells are not cryoprotected prior to stimulation, and the duration or amount of time required to complete the provided process, measured from isolation, enrichment, and/or selection of the input cells to the time the output cells are harvested, formulated, and/or cryoprotected, is about 48 hours, 72 hours, 96 hours, or 120 hours, or is less than 48 hours, 72 hours, 96 hours, or 120 hours.

In certain embodiments, the provided processes are performed on a population of cells, such as CD4+ and CD8+ T cells or CD3+ T cells, that have been isolated, enriched, or selected from a biological sample. In some aspects, the provided methods can generate or produce compositions of engineered T cells within a reduced amount of time from when the biological sample is collected from a subject compared to other methods or processes. In some embodiments, the provided methods can generate or produce engineered T cells within or about 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, or within 120 hours, 96 hours, 72 hours, or 48 hours from when the biological sample is collected from the subject to when the engineered T cells are collected, harvested, or formulated (e.g., for cryoprotection or administration), including any or all times that the biological sample, or enriched, isolated, or selected cells are cryopreserved and stored prior to the step for transduction. In some embodiments, the provided methods can generate or produce engineered T cells within 5 days, or within about 5 days, or within about 4 days from when the biological sample is collected from the subject to when the engineered T cells are collected, harvested, or formulated (e.g., for cryoprotection or administration), including any or all times that the biological sample, or enriched, isolated, or selected cells are cryopreserved and stored prior to the step for transduction.

In certain embodiments, the provided methods are used in conjunction with a process for generating or producing an output population of output cells and/or enriched T cells. In particular embodiments, the output population of output cells and/or enriched T cells is or includes cells collected, obtained, isolated, selected and/or enriched from a biological sample, such as a blood sample or a leukapheresis sample; cells incubated under stimulatory conditions; cells engineered, e.g., transduced, to express or contain a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor, e.g., a CAR; cells cultured to a threshold amount, threshold density, or threshold expansion culture; and/or formulated cells. In some embodiments, the cells of the output population are cryoprotected and thawed, e.g., during, prior to, and/or after one or more steps of the process. In some embodiments, the output population (e.g., a therapeutic cell composition) contains T cells, e.g., CD4+ T cells and CD8+ T cells, expressing a recombinant receptor, e.g., a CAR.

In some embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, at least 95% of the cells of the output population (e.g., therapeutic cell composition) express the recombinant receptor. In certain embodiments, at least 50% of the cells of the output composition express the recombinant receptor. In certain embodiments, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD3+ T cells of the output composition (e.g., therapeutic cell composition) express the recombinant receptor. In some embodiments, at least 50% of the CD3+ T cells of the output composition express the recombinant receptor. In particular embodiments, at least at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or greater than 99% of the CD4+ T cells of the output composition (e.g., therapeutic cell composition) express the recombinant receptor. In particular embodiments, at least 50% of the CD4+ T cells of the output composition (e.g., therapeutic cell composition) express the recombinant receptor. In some embodiments, at least at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more than 99% of the CD8+ T cells of the output composition (e.g., therapeutic cell composition) express the recombinant receptor. In certain embodiments, at least 50% of the CD8+ T cells of the output composition (e.g., therapeutic cell composition) express the recombinant receptor.

In certain embodiments, the cells of the output composition (e.g., a therapeutic cell composition) have enhanced cytolytic activity against cells expressing an antigen bound and/or recognized by the recombinant receptor (e.g., target cells) compared to output cells produced by an alternative process, e.g., a process including one or more steps of expanding the cells. In some embodiments, when cells of the output composition (e.g., a therapeutic cell composition) are exposed to cells expressing an antigen, e.g., target cells, the cells of the output composition kill about or at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells expressing the antigen. In certain embodiments, the cells of the output composition (e.g., a therapeutic cell composition) kill at least 25%, 50%, 75%, 100%, 150%, or 1-, 2-, 3-, 4-, or 5-fold more antigen-expressing cells, e.g., target cells, than output cells produced by an alternative process under similar or comparable conditions.

In certain embodiments, the cells of the output population (e.g., a therapeutic cell composition) have improved anti-tumor activity in vivo when compared to output cells produced by an alternative process, e.g., a process that includes one or more steps of expanding the cells. In some embodiments, when the cells of the output composition (e.g., a therapeutic cell composition) are administered to a subject, e.g., a subject having a tumor or cancer, the cells of the output population kill about or at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of tumor cells, e.g., cancer or tumor cells expressing the antigen, in the subject. In certain embodiments, the cells of the output composition (e.g., a therapeutic cell composition) kill at least 25%, 50%, 75%, 100%, 150%, or 1-, 2-, 3-, 4-, or 5-fold more tumor cells in vivo than output cells produced by an alternative process under similar or comparable conditions.

In particular embodiments, the majority of the cells of the output population (e.g., a therapeutic cell composition) are naive-like cells, central memory cells, and/or effector memory cells. In particular embodiments, the majority of the cells of the output population (e.g., a therapeutic cell composition) are naive-like cells or central memory cells. In some embodiments, the majority of the cells of the output population (e.g., a therapeutic cell composition) are positive for one or more of CCR7 expression or CD27 expression. In certain embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a greater amount of naive-like cells or central memory cells than output populations generated from alternative processes, such as processes involving expansion.

In certain embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a low quantity and/or frequency of exhausted and/or senescent cells. In particular embodiments, the cells of the output population have a low quantity and/or frequency of exhausted and/or senescent cells. In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of the output population (e.g., a therapeutic cell composition) are exhausted and/or senescent. In certain embodiments, less than 25% of the cells of the output population (e.g., a therapeutic cell composition) are exhausted and/or senescent. In certain embodiments, less than 10% of the cells of the output population are exhausted and/or senescent. In particular embodiments, the cells have a low quantity.

In some embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a low quantity and/or frequency of cells that are negative for CD27 expression, e.g., surface expression, and CCR7 expression. In particular embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a low quantity and/or frequency of CD27-CCR7- cells. In some embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of the output population (e.g., a therapeutic cell composition) are CD27-CCR7- cells. In certain embodiments, less than 25% of the cells of the output population are CD27-CCR7- cells. In certain embodiments, less than 10% of the cells of the output population are CD27-CCR7- cells. In embodiments, less than 5% of the cells of the output population are CD27-CCR7- cells.

In some embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a high quantity and/or frequency of cells that are positive for one or both of CD27 and CCR7 expression, e.g., surface expression. In some embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a high quantity and/or frequency of cells that are positive for one or both of CD27 and CCR7. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the cells of the output population are positive for one or both of CD27 and CCR7. In various embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD4+CAR+ cells of the output population (e.g., a therapeutic cell composition) are positive for one or both of CD27 and CCR7. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the CD8+ CAR+ cells of the output population (e.g., therapeutic cell composition) are positive for one or both of CD27 and CCR7.

In certain embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a high quantity and/or frequency of cells that are positive for CD27 expression and CCR7 expression, e.g., surface expression. In some embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a high quantity and/or frequency of CD27+CCR7+ cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the cells of the output population (e.g., a therapeutic cell composition) are CD27+CCR7+ cells. In various embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the CD4+CAR+ cells of the output population (e.g., a therapeutic cell composition) are CD27+CCR7+ cells. In some embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the CD8+ CAR+ cells of the output population (e.g., therapeutic cell composition) are CD27+ CCR7+ cells.

In certain embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a low quantity and/or frequency of cells that are negative for CCR7 expression, e.g., surface expression, and positive for CD45RA expression. In some embodiments, the cells of the output population (e.g., a therapeutic cell composition) have a low quantity and/or frequency of CCR7-CD45RA+ cells. In particular embodiments, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the cells of the output population (e.g., a therapeutic cell composition) are CCR7-CD45RA+ cells. In some embodiments, less than 25% of the cells of the output population (e.g., a therapeutic cell composition) are CCR7-CD45RA+ cells. In particular embodiments, less than 10% of the cells of the output population (e.g., a therapeutic cell composition) are CCR7-CD45RA+ cells. In certain embodiments, less than 5% of the cells in the output population (e.g., therapeutic cell composition) are CCR7-CD45RA+ cells.

In any of the above embodiments, a selection step (e.g., a polishing step; see Sections I-I) can be used to achieve a fraction, frequency, concentration and/or percentage of cells of a particular phenotype or function in an output population (e.g., a therapeutic cell composition). In any of the above embodiments, a selection step (e.g., a polishing step; see Sections I-I) can be used to select for a desired population.

In certain embodiments, the cells are harvested prior to 1, 2, 3, 4, 5, 6, 8, 10, 20, or more cell doublings of the cell population, such as doublings that occur during incubation, prior to about 1, 2, 3, 4, 5, 6, 8, 10, 20, or more cell doublings, or prior to at least 1, 2, 3, 4, 5, 6, 8, 10, 20, or more cell doublings. In certain embodiments, the cells are harvested prior to any doubling of the population, such as doublings that occur during incubation. In some aspects, decreasing the doublings that may occur during the engineering process increases, in some embodiments, the portion of engineered T cells that are naive strains. In some embodiments, increasing the doublings during the engineering process increases the differentiation of T cells that may occur during the engineering process.

In some aspects, for a process for generating or producing an engineered cell composition (e.g., a therapeutic cell composition), it is contemplated that decreasing the expansion or cell doublings that occur during the process, e.g., during incubation, increases the amount or portion of naive-like T cells in the resulting engineered cell composition. In certain aspects, increasing the expansion or cell doublings that occur during the process increases the amount or portion of differentiated T cells in the resulting engineered cell composition. In some aspects, it is contemplated that processes such as those provided herein that increase or expand the portion of naive-like cells in the resulting engineered cell composition may increase the efficacy, potency, and persistence of the engineered cell composition, e.g., in vivo after administration.

II. Agent and Reagent System In certain aspects, the method uses a reversible system in which at least one agent (e.g., a selection agent or a stimulating agent) capable of binding to a molecule on the surface of a cell (a cell surface molecule) reversibly associates with a reagent (e.g., a selection agent or a stimulating agent). In some cases, the reagent contains multiple binding sites capable of reversibly binding to the agent (e.g., a selection agent or a stimulating agent). In some cases, the reagent (e.g., a selection agent or a stimulating agent) is a multimerization reagent. In some embodiments, at least one agent (e.g., a selection agent or a stimulating agent) contains at least one binding site B that can specifically bind to an epitope or a region of a molecule, and also contains a binding partner C that specifically binds to at least one binding site Z of the reagent (e.g., a selection agent or a stimulating agent). In some cases, the binding interaction between the binding partner C and at least one binding site Z is a non-covalent interaction. In some embodiments, the binding interaction, such as a non-covalent interaction, between the binding partner C and at least one binding site Z is reversible.

In some embodiments, the reversible association can be mediated in the presence of a substance, such as a competitor or free binder, that is or contains a binding site that is also capable of binding to at least one binding site Z. In general, the substance (e.g., competitor or free binder) can act as a competitor by virtue of its higher binding affinity for the binding site Z present in the reagent than the binding partner C and/or by virtue of its presence in a higher concentration than the binding partner C, thereby dissociating and/or dissociating the binding partner C from the reagent. In some embodiments, the substance (e.g., competitor or free binder) for at least one binding site Z is greater than the affinity of the binding partner C of the agent (e.g., selection agent or stimulant) for at least one binding site Z. Thus, in some cases, the binding between the binding site Z of the reagent and the binding partner C of the agent (e.g., selection agent or stimulant) can be disrupted by the addition of a substance (e.g., competitor or free binding partner). This makes the association of the agent (e.g., a selection agent or stimulant) and the reagent (e.g., a selection agent or stimulant reagent) reversible.

Reagents that can be used in such reversible systems are described and known in the art, see, e.g., U.S. Pat. Nos. 5,168,049, 5,506,121, 6,103,493, 7,776,562, 7,981,632, 8,298,782, 8,735,540, 9,023,604, and published PCT applications WO2013/124474 and WO2014/076277. Non-limiting examples of reagents and binding partners capable of forming reversible interactions, as well as substances capable of reversing such binding (e.g., competitors or free binders), are described below.

A. Reagents Reagents contemplated herein include selection and stimulating reagents. In some embodiments, the selection and stimulating reagents are the same. In some embodiments, the selection and stimulating reagents are different. However, both the selection and stimulating reagents may be formulated from the same material. In some embodiments, the reagent (e.g., selection or stimulating reagent) contains one or more binding sites Z that are capable of reversibly binding to a binding partner C contained in an agent (e.g., selection or stimulating agent). In some embodiments, the reagent contains multiple binding sites Z, each of which can specifically bind to a binding partner C contained in an agent (e.g., selection or stimulating agent), such that the reagent has the ability to reversibly bind to multiple agents (e.g., selection or stimulating agents), such as a multimerization reagent (e.g., selection or stimulating reagent). In some embodiments, the reagent is an oligomer, or a polymer of individual molecules (e.g., monomers) or a complex (e.g., tetramer) that is composed of individual molecules, each of which contains at least one binding site Z. In some embodiments, the reagent contains at least two binding sites Z, at least three binding sites Z, at least four binding sites Z, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or more binding sites Z. The binding sites can all be the same, or the multiple binding sites can contain one or more different binding sites (e.g., Z1, Z2, Z3, etc.).

In some embodiments, two or more agents (e.g., selection agents or stimuli) are associated, e.g., reversibly bound to the agent (e.g., selection agent or stimuli) via one or more binding sites Z present on the agent (e.g., selection agent or stimuli). In some cases, this results in the agents (e.g., selection agents or stimuli) being closely spaced such that an avidity effect can occur when a target cell having (at least two copies of) a cell surface molecule is contacted with an agent (e.g., selection agent or stimuli) having one or more binding sites B that can bind to that particular molecule.

In some embodiments, two or more different agents (e.g., selection agents or stimuli) that are the same, i.e., contain the same binding site B, can be reversibly bound to the reagent. In some embodiments, it is possible to use at least two different (types) agents (e.g., selection agents or stimuli), and possibly three or four different (types) agents, such as, for example, two or more different selection agents and/or stimuli. For example, in some embodiments, the reagent (e.g., selection agent or stimuli reagent) can be reversibly bound to a first agent (e.g., selection agent or stimuli) that contains binding site B1, B2, B3, or B4, and a second agent (e.g., selection agent or stimuli) that contains another binding site, such as, for example, another of binding sites B1, B2, B3, or B4. In some cases, the binding sites of the first agent and the second agent can be the same. For example, in some aspects, each of the at least two agents (e.g., selection agents or stimuli) can bind to the same molecule. In some cases, the binding sites of the first and second agents can be different. In some aspects, each of at least two agents (e.g., selection agents or stimuli) can bind to different molecules, e.g., the first molecule, the second molecule, etc. In some cases, the different molecules, e.g., different cell surface molecules, can be present on the same target cell. In another example, the different molecules, e.g., different cell surface molecules, can be present on different target cells present in the same population of cells. In some cases, a third, fourth, etc. agent (e.g., selection agent or stimuli) can be associated with the same reagent (e.g., selection reagent or stimuli reagent), each of which further contains a different binding site.

In some embodiments, two or more different agents (e.g., selection agents or stimulants) contain the same binding partner C. In some embodiments, two or more different agents (e.g., selection agents or stimulants) contain different binding partners. In some aspects, a first agent (e.g., selection agent or stimulant) can have a binding partner C1 that can specifically bind to a binding site Z1 present on a reagent (e.g., selection agent or stimulant), and a second agent (e.g., selection agent or stimulant) can have a binding partner C2 that can specifically bind to a binding site Z1 or a binding site Z2 present on a reagent (e.g., selection agent or stimulant). Thus, in some examples, the multiple binding sites Z contained in the reagent include binding sites Z1 and Z2, which are capable of reversibly binding to binding partners C1 and C2, respectively, contained in the agent (e.g., selection agent or stimulant). In some embodiments, C1 and C2 are the same, and/or Z1 and Z2 are the same. In another aspect, one or more of the multiple binding sites Z can be different. In another example, one or more of the multiple binding partners C may be different. It is within the level of one of skill in the art to choose any combination of different binding partners C that is compatible with a reagent containing binding site Z, so long as each of the binding partners C is capable of interacting with, e.g., specifically binding to, one of the binding sites Z.

In some embodiments, the reagent (e.g., the selection reagent or the stimulating reagent) is streptavidin, a streptavidin mutein or the like, avidin, an avidin mutein or an analog thereof (neutravidin), or a mixture thereof, and such reagent contains one or more binding sites Z that reversibly associate with a binding partner C. In some embodiments, the binding partner C can be biotin, a biotin derivative or an analog thereof, or a streptavidin-binding peptide or another molecule that can specifically bind to streptavidin, a streptavidin mutein or an analog thereof, avidin or an avidin mutein or an analog thereof. In some embodiments, the reagent is or contains streptavidin, avidin, an analog or mutein of streptavidin, or an analog or mutein of avidin that reversibly binds to biotin, a biotin analog, or a biologically active fragment thereof. In some embodiments, the reagent (e.g., the selection or stimulating reagent) is or contains a streptavidin analog or mutein, or an avidin analog or mutein that reversibly binds to a streptavidin-binding peptide. In some embodiments, the substance (e.g., the competitor or free binder) can be biotin, a biotin derivative or biotin analog, or a streptavidin-binding peptide that has the ability to compete for binding with a binding partner C for one or more binding sites Z. In some embodiments, the binding partner C and the substance (e.g., the competitor and/or free binder) are different, and the substance (e.g., the competitor and/or free binder) exhibits a higher binding affinity for one or more binding sites Z compared to the affinity of the binding partner.

In some embodiments, the streptavidin can be wild-type streptavidin, a streptavidin mutein, such as a streptavidin-like polypeptide, or an analog thereof. Similarly, the avidin in some aspects includes a mutein or analog of avidin, such as wild-type avidin or neutravidin, deglycosylated avidin with a modified arginine that typically exhibits a greater neutral charge and can be used as an alternative to native avidin. In general, deglycosylated neutral avidin includes commercially available forms, such as "Extravidin" available from Sigma Aldrich, or "Neutravidin" available from Thermo Scientific or Invitrogen.

In some embodiments, the reagent (e.g., selection or stimulation reagent) is streptavidin or a streptavidin mutein or analog thereof. In some embodiments, wild-type streptavidin (wt-streptavidin) has the amino acid sequence disclosed in Argarana et al., Nucleic Acids Res. 14 (1986) 1871-1882 (SEQ ID NO: 1). Generally, streptavidin occurs naturally as a tetramer of four identical subunits, i.e., a homotetramer, where each subunit contains a single binding site for biotin, a biotin derivative or analog thereof, or a biotin mimetic. An exemplary sequence of a subunit of streptavidin is the sequence of amino acids shown in SEQ ID NO: 1, but such a sequence may also include sequences present in its homologs from other Streptomyces species. In particular, each subunit of streptavidin can exhibit strong binding affinity for biotin, with an equilibrium dissociation constant (K _D ) on the order of approximately 10 ⁻¹⁴ M. In some cases, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al. (2006) Nat. Methods, 3:267-73; Zhang et al. (2015) Biochem. Biophys. Res. Commun., 463:1059-63), as a bivalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. Biol., 426:199-214), or in monomeric or dimeric forms (Wu et al. (2005) J. Biol. Chem., 280:23225-31; Lim et al. (2010) Biochemistry, 50:8682-91).

In some embodiments, the streptavidin may be in any form, such as wild-type or unmodified streptavidin, such as streptavidin from Streptomyces species, which generally contains at least one functional subunit of wild-type streptavidin from Streptomyces avidinii as shown in SEQ ID NO:1, or a functionally active fragment thereof, which contains at least one functional subunit that contains a binding site for biotin, a biotin derivative or analog thereof, or a biotin mimetic. For example, in some embodiments, the streptavidin may include a fragment of wild-type streptavidin that is truncated at the N-terminus and/or C-terminus. Such minimal streptavidins include any that start at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO:1 and end at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO:1. In some embodiments, a functionally active fragment of streptavidin contains a sequence of amino acids set forth in SEQ ID NO:2. In some embodiments, a functionally active fragment of streptavidin contains a sequence of amino acids set forth in SEQ ID NO:103. In some embodiments, the streptavidin set forth in SEQ ID NO:2 may further contain an N-terminal methionine at a position corresponding to Ala13 with the numbering set forth in SEQ ID NO:1. In some embodiments, the functionally active fragment of streptavidin set forth in SEQ ID NO:103 does not have an N-terminal methionine at a position corresponding to Ala13 with the numbering set forth in SEQ ID NO:1. References to residue positions in streptavidin or streptavidin muteins are with reference to the numbering of residues in SEQ ID NO:1.

In some aspects, a streptavidin mutein comprises a polypeptide that is distinct from the sequence of unmodified streptavidin or wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but that comprises at least one functional subunit that contains a binding site for biotin, a biotin derivative or analog thereof, or a streptavidin-binding peptide. In some aspects, a streptavidin-like polypeptide and a streptavidin mutein can be a polypeptide that is essentially immunologically equivalent to wild-type streptavidin, and in particular has the ability to bind biotin, a biotin derivative, or a biotin analog with the same or different affinity as wt-streptavidin. In some cases, a streptavidin-like polypeptide or a streptavidin mutein may contain amino acids that are not part of wild-type streptavidin, or may include only a portion of wild-type streptavidin. In some embodiments, streptavidin-like polypeptide is a polypeptide that is not identical to wild-type streptavidin. This is because the host does not have the enzyme required to transform the host-produced polypeptide into the structure of wild-type streptavidin. In some embodiments, streptavidin can also exist as streptavidin tetramers and streptavidin dimers, particularly streptavidin homotetramers, streptavidin homodimers, streptavidin heterotetramers and streptavidin heterodimers. In general, each subunit usually has a binding site for biotin or biotin analogs, or for streptavidin-binding peptides. Examples of streptavidin or streptavidin muteins are mentioned, for example, in WO 86/02077, DE 19641876 A1, U.S. Pat. No. 6,022,951, WO 98/40396 or WO 96/24606.

In some embodiments, a streptavidin mutein can have amino acids that are not part of unmodified or wild-type streptavidin or can contain only a portion of wild-type or unmodified streptavidin. In some embodiments, a streptavidin mutein contains at least one subunit that can have one or more amino acid substitutions (replacements) when compared to a subunit of unmodified or wild-type streptavidin, e.g., as shown in SEQ ID NO:1 or a functionally active fragment thereof, e.g., as shown in SEQ ID NO:2 or SEQ ID NO:103. In some embodiments, at least one subunit of a streptavidin mutein may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid differences compared to wild-type streptavidin or unmodified streptavidin and/or contains at least one subunit comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of amino acids set forth in SEQ ID NO:1, 2 or 103, when such streptavidin mutein exhibits functional activity of binding biotin, a biotin derivative or analogue thereof, or a biotin mimetic. In some embodiments, the amino acid substitutions are conservative or non-conservative. Examples of streptavidin muteins are known in the art. See, for example, U.S. Patent Nos. 5,168,049; 5,506,121; 6,022,951; 6,156,493; 6,165,750; 6,103,493; or 6,368,813; or published PCT application WO2014/076277.

In some embodiments, the streptavidin or streptavidin mutein comprises a protein containing one or more functional subunits that contain one or more binding sites Z for biotin, a biotin derivative or analog, or a streptavidin-binding peptide, e.g., two or more, three or more, four or more, and possibly five, six, seven, eight, nine, ten, eleven, twelve or more functional subunits. In some embodiments, the streptavidin or streptavidin mutein can comprise a monomer; a dimer, including a heterodimer or homodimer; a tetramer, including a homotetramer, a heterotetramer, a monovalent tetramer, or a divalent tetramer; or a higher order multimer or oligomer thereof.

In some embodiments, the binding affinity of streptavidin or streptavidin muteins for peptide ligand binding partners is less than 1×10 ⁻⁴ M, less than 5×10 ⁻⁴ M, less than 1×10 ⁻⁵ M, less than 5× ^{10 −5} M, less than 1×10 −6 M, less than 5×10 ⁻⁶ M or less than 1×10 ⁻⁷ M, but typically greater than 1×10 ⁻¹³ M, greater than 1×10 ⁻¹² M or greater than 1×10 ⁻¹¹ M. For example, peptide sequences such as those disclosed in U.S. Pat. No. 5,506,121 (Strep-tag) act as biotin mimics and demonstrate binding affinity for streptavidin, e.g., with a K _D of about 10 ⁻⁴ M to 10 ⁻⁵ M. In some cases, binding affinity can be further improved by making mutations within the streptavidin molecule. See, e.g., U.S. Patent No. 6,103,493 or published PCT application WO2014/076277. In some embodiments, binding affinity can be measured by methods known in the art, such as any of those described below.

In some embodiments, a reagent (e.g., a selection or stimulating agent), such as streptavidin or a streptavidin mutein, exhibits binding affinity for a peptide ligand binding partner. The peptide ligand binding partner can be binding partner C present in the agent (e.g., a selection or stimulating agent). In some embodiments, the peptide sequence contains a sequence of the general formula shown in SEQ ID NO:9, e.g., a sequence shown in SEQ ID NO:10. In some embodiments, the peptide sequence has the general formula shown in SEQ ID NO:11, e.g., the general formula shown in SEQ ID NO:12. In some examples, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag® and shown in SEQ ID NO:7). In some examples, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II and shown in SEQ ID NO:8). In some embodiments, the peptide ligand contains a contiguous sequence of at least two streptavidin binding modules, the distance between the two modules being at least 0 amino acids and less than 50 amino acids, one binding module having 3-8 amino acids and containing at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine or methionine. Another binding module is the same streptavidin peptide ligand as shown in SEQ ID NO:11 or a different streptavidin peptide ligand (see, e.g., International Published PCT Application WO02/077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand contains a sequence having a formula shown in any of SEQ ID NOs:13 or 14. In some embodiments, the peptide ligand has a sequence of amino acids shown in any of SEQ ID NOs:15-19.

In some embodiments, the reagent (e.g., the selection reagent or the stimulation reagent) is or contains a streptavidin mutein. In some embodiments, the streptavidin mutein contains one or more mutations (e.g., amino acid substitutions) compared to the wild-type streptavidin shown in SEQ ID NO:1 or a biologically active portion thereof (e.g., SEQ ID NO:2 or SEQ ID NO:103). For example, the biologically active portion of streptavidin can include a streptavidin variant that is truncated at the N-terminus and/or C-terminus, sometimes referred to as a minimal streptavidin. In some embodiments, any of the mutations made therein include an N-terminally truncated minimal streptavidin that starts at the N-terminus in the region of amino acid positions 10-16 and ends at the C-terminus in the region of amino acid positions 133-142, compared to the sequence shown in SEQ ID NO:1. In some embodiments, the minimal streptavidin, truncated at the N-terminus and into which any mutations have been made, contains the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the minimal streptavidin contains the amino acid sequence from position Ala13 to Ser139, optionally with an N-terminal methionine residue in place of Ala13. For purposes herein, the numbering of the amino acid positions refers generally to the numbering of wt-streptavidin shown in SEQ ID NO:1 (see, e.g., Argarana et al., Nucleic Acids Res. 14 (1986), 1871-1882, also FIG. 3).

In some embodiments, the streptavidin mutein is a mutant described in U.S. Patent No. 6,103,493. In some embodiments, the streptavidin mutein contains at least one mutation within the region of amino acid positions 44-53 based on the amino acid sequence of wild-type streptavidin as shown in SEQ ID NO:1. In some embodiments, the streptavidin mutein contains mutations at one or more residues 44, 45, 46 and/or 47. In some embodiments, the streptavidin mutein contains a substitution of an aliphatic amino acid, such as, for example, Glu at position 44 of wild-type streptavidin with a hydrophobic aliphatic amino acid, such as Val, Ala, Ile or Leu, any amino acid at position 45, a hydrophobic aliphatic amino acid at position 46, and/or a substitution of Val at position 47 with a basic amino acid, such as, for example, Arg or Lys, typically Arg. In some embodiments, Ala is at position 46, and/or Arg is at position 47, and/or Val or Ile is at position 44. In some embodiments, the streptavidin mutant contains residues Val 44 -Thr 45 -Ala 46 -Arg 47 as shown in the exemplary streptavidin mutein containing the sequence of amino acids set forth in SEQ ID NO:3, SEQ ID NO:4 (also known as streptavidin mutation 1, SAM1), or SEQ ID NO:104. In some embodiments, the streptavidin mutein contains residues Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ as shown in the exemplary streptavidin containing the sequence of amino acids set forth in SEQ ID NO ^{: 5} , SEQ ID NO: ⁶ (also known as SAM2), or SEQ ID NO: ¹⁰⁵ . In some cases, such streptavidin muteins are described, for example, in US Pat. No. 6,103,493 and are commercially available under the registered trademark Strep-Tactin®.

In some embodiments, the streptavidin mutein is a variant described in International Published PCT Application WO2014/076277. In some embodiments, the streptavidin mutein contains at least two cysteine residues in the region of amino acid positions 44-53, in accordance with the amino acid positions set forth in SEQ ID NO:1. In some embodiments, cysteine residues are present at positions 45 and 52 to generate disulfide bridge connections with these amino acids. In such embodiments, amino acid 44 is typically glycine or alanine, amino acid 46 is typically alanine or glycine, and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutein contains at least one mutation or amino acid difference in the region of amino acid residues 115-121, in accordance with the amino acid positions set forth in SEQ ID NO:1. In some embodiments, the streptavidin mutein contains at least one mutation at amino acid positions 117, 120, and 121, and/or a deletion of amino acids 118 and 119 and a substitution at least at amino acid position 121.

In some embodiments, the streptavidin mutein contains a mutation at a position corresponding to position 117, which can be to a large hydrophobic residue such as Trp, Tyr or Phe, or a charged residue such as Glu, Asp or Arg, or a hydrophilic residue such as Asn or Gin, or optionally to the hydrophobic residues Leu, Met or Ala, or the polar residues Thr, Ser or His. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120, which can be to a small residue such as Ser or Ala or Gly, and a mutation at a position corresponding to position 121, which can be to a hydrophobic residue such as a bulky hydrophobic residue such as Trp, Tyr or Phe. In some embodiments, the mutation at position 117 is combined with a mutation at a position corresponding to position 120 of wild type streptavidin or a biologically active fragment thereof shown in SEQ ID NO:1, which may be to a hydrophobic mutation such as Leu, Ile, Met or Val, or generally Tyr or Phe, and a mutation at a position corresponding to position 121 compared to the position of wild type streptavidin or a biologically active fragment thereof shown in SEQ ID NO:1, which may be to a small residue such as Gly, Ala or Ser, or combined with Gln, or combined with a hydrophobic residue such as Leu, Val, Ile, Trp, Tyr, Phe or Met. In some embodiments, such muteins may also contain residues Val44-Thr45-Ala46-Arg47 or residues Ile44-Gly45-Ala46-Arg47. In some embodiments, the streptavidin mutein contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121. In some embodiments, the mutein streptavidin contains the sequence of amino acids set forth in SEQ ID NO:27 or SEQ ID NO:28, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in SEQ ID NO:27 or SEQ ID NO:28, contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121, and exhibits functional activity of binding to biotin, a biotin analog or a streptavidin-binding peptide.

In some embodiments, the streptavidin mutein can contain any of the above mutations in any combination, and the resulting streptavidin mutein has a cytotoxicity of less than 2.7×10 M for the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also known as Strep-tag® and shown in SEQ ID NO:7) and/or less than 1.4× ¹⁰ M for the peptide ligand (Trp-Ser-His ^{-Pro-Gln-Phe-Glu-Lys; also known as Strep-tag® II and shown in SEQ ID NO:8) and/or less than 1×10 M} , 5 ^{× 10} M, 1× ¹⁰ M, 5× ¹⁰ M, 1× ¹⁰ M, 5× ¹⁰ M or 1× ¹⁰ M, but less than 1×10 M, ... The antibody may exhibit a binding affinity generally greater than ^-12 M or 1 x 10 ^-11 M.

In some embodiments, the streptavidin mutein exhibits a sequence of amino acids set forth in any of SEQ ID NOs:3-6, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:104 or SEQ ID NO:105, or a sequence of amino acids exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of amino acids set forth in any of SEQ ID NOs:3-6, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:104 or SEQ ID NO:105, and has a molecular weight of less than 2.7×10 −4 M to the peptide ligand (Trp Arg His Pro Gln Phe Gly Gly; also known as Strep-tag® and set forth in SEQ ID NO:7), and/or a molecular weight of less than 2.7×10 ⁻⁴ M to the peptide ligand (Trp Ser His Pro Gln Phe Glu Lys; also known as Strep-tag (registered trademark) II and shown in SEQ ID NO:8) and/or less than 1×10 ⁻⁴ M, 5×10 ⁻⁴ M, 1×10 ⁻⁵ M, 5×10 ⁻⁵ M, 1×10 ⁻⁶ M, 5×10 ⁻⁶ M or 1×10 ⁻⁷ M, but typically greater than 1×10 ⁻¹³ M, 1×10 ⁻¹² M or 1× ^{10 −11 M} to any peptide ligand shown in any of SEQ ID NOs:7-19.

In some embodiments, the streptavidin mutein also exhibits binding to another streptavidin ligand, such as, but not limited to, biotin, iminobiotin, lipoic acid, desthiobiotin, diaminebiotin, HABA (hydroxyazobenzene-benzoic acid) and/or dimethyl-HABA. In some embodiments, the streptavidin mutein exhibits a binding affinity for another streptavidin ligand, such as biotin or desthiobiotin, that is greater than the binding affinity of the streptavidin mutein for a biotin mimetic peptide ligand, such as, for example, any of SEQ ID NOs:7-19. Thus, in some embodiments, biotin or a biotin analog or derivative (e.g., desthiobiotin) may be used as a competitor in the methods provided. For example, a mutein streptavidin designated Strep-tactin® (e.g., containing the sequence shown in SEQ ID NO:4) having a peptide ligand designated Strep-tag® II (e.g., as shown in SEQ ID NO:8) is characterized by a binding affinity with a K _D of about 10 ⁻⁶ M, as compared to a K _D of about 10 ⁻¹³ M for the biotin-streptavidin interaction. In some cases, biotin, which can bind with high affinity to Strep-tactin® with a K _D of 10 ⁻¹⁰ to 10 ⁻¹³ M or about 10 ⁻¹⁰ to 10 ⁻¹³ M, may compete with Strep-tag® II for the binding site.

In some cases, the reagent (e.g., the selection reagent or the stimulating reagent) contains at least two chelating groups K that may be capable of binding to a transition metal ion. In some embodiments, the reagent (e.g., the selection reagent or the stimulating reagent) may be capable of binding to an oligohistidine affinity tag, glutathione-S-transferase, calmodulin or an analog thereof, calmodulin-binding peptide (CBP), FLAG-peptide, HA tag, maltose-binding protein (MBP), an HSV epitope, a myc epitope, and/or a biotinylated carrier protein.

In some embodiments, the reagent (e.g., the selection or stimulating reagent) is an oligomer or polymer. In some embodiments, the oligomer or polymer can be generated by either directly or indirectly binding to individual molecules of naturally occurring proteins, directly or indirectly binding to individual molecules of monomers or complexes of subunits that make up the individual molecules (e.g., directly or indirectly binding to dimers, trimers, tetramers of naturally occurring proteins). For example, homodimers or heterodimers of streptavidin or avidin tetramers may be referred to as individual molecules or the smallest basic units of the respective oligomers or polymers. In some embodiments, the oligomer or polymer can contain an association of at least two individual molecules of the protein (e.g., a dimer) or can be at least a trimer, 4mer, 5mer, 6mer, 7mer, 8mer, 9mer, 10mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer, or 50-mer of individual molecules of the protein (e.g., a monomer, a tetramer).

Oligomers can be produced using any method known in the art, such as, for example, any of those described in U.S. Patent Application Publication No. 2004/0082012. In some embodiments, the oligomer or polymer contains two or more individual molecules that can be crosslinked, such as by a polysaccharide or bifunctional linker.

In some embodiments, oligomers or polymers are obtained by crosslinking individual molecules or complexes of subunits constituting individual molecules in the presence of a polysaccharide. In some embodiments, oligomers or polymers can be prepared by introducing carboxyl residues into a polysaccharide, e.g., dextran. In some aspects, individual molecules of reagent (e.g., monomers, tetramers) can be coupled to carboxyl groups in the dextran backbone via primary amino groups of internal lysine residues and/or the free N-terminus using conventional carbodiimide chemistry. In some embodiments, the coupling reaction is carried out at a molar ratio of individual molecules of reagent (e.g., monomers, tetramers) of about 60 mol per mol of dextran.

In some embodiments, the reagent (e.g., selection or stimulating reagent) is an oligomer or polymer of one or more streptavidins (e.g., Strep-Tactin® or Strep-Tactin® XT), or avidin or any analog or mutein of avidin (e.g., neutravidin). In some embodiments, the binding site Z is the native biotin binding site of avidin or streptavidin, for which there may be up to four binding sites in an individual molecule (e.g., a tetramer contains four binding sites Z). Thus, a homotetramer may contain up to four identical binding sites, i.e., Z1, while a heterotetramer may contain four binding sites that may be different, e.g., containing Z1 and Z2. In some embodiments, the oligomer is generated or made from multiple individual molecules (e.g., multiple homotetramers) of the same streptavidin, streptavidin mutein, avidin, or avidin mutein, where each binding site Z, e.g., Z, of the oligomer is the same. For example, in some cases, the oligomer can contain multiple binding sites Z, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more binding sites Z. In some embodiments, the oligomer is generated or produced from a plurality of individual molecules which may be heterotetramers of streptavidin, streptavidin muteins, avidin or avidin muteins, and/or two or more different individual molecules (e.g., different homotetramers) of streptavidin, streptavidin muteins, avidin or avidin muteins which differ in their binding sites Z, e.g., Z1 and Z2, in which case a plurality of different binding sites Z, e.g., Z1 and Z2, may be present within the oligomer. For example, in some cases, an oligomer may contain multiple binding sites Z, which may include multiple binding sites Z1 and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more combined binding sites Z1 and Z2.

Optionally, the respective oligomer or polymer may be crosslinked by a polysaccharide. In one embodiment, an oligomer or polymer of streptavidin, or avidin, or an analog of streptavidin, or an analog of avidin (e.g., neutravidin) may be prepared in a first step by introducing carboxyl residues into a polysaccharide, such as dextran, essentially as described in Noguchi, A, et al, Bioconjugate Chemistry (1992) 3, 132-137. In some such aspects, the streptavidin or avidin or analog may then be linked in a second step via the primary amino groups of internal lysine residues and/or the free N-terminus to the carboxyl groups of the dextran backbone using conventional carbodiimide chemistry. In some cases, crosslinked oligomers or polymers of streptavidin or avidin or analogs of streptavidin or avidin can be obtained by crosslinking via bifunctional molecules that act as linkers, such as glutaraldehyde, or by other methods described in the art.

In some embodiments, oligomers or polymers are obtained by crosslinking the individual molecules or complexes of subunits that make up the individual molecules using bifunctional linkers or other chemical linkers such as glutaraldehyde, or by other methods known in the art. In some aspects, crosslinked oligomers or polymers of streptavidin or avidin or any mutein of streptavidin or avidin, or analogs of streptavidin or avidin, can be obtained by crosslinking the individual streptavidin or avidin molecules via a bifunctional molecule that acts as a linker, such as glutaraldehyde, or by other methods described in the art. For example, it is possible to generate oligomers of streptavidin muteins by introducing thiol groups into the streptavidin muteins (this can be done, for example, by reacting streptavidin with 2-iminothiolane (Trauts' reagent) and activating, for example in a separate reaction, the amino groups available on the streptavidin muteins). In some embodiments, such activation of amino groups can be achieved by reacting the streptavidin mutein with commercially available heterobifunctional cross-linkers such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or succinimidyl-6-[(β-maleimidopropionamido)hexanoate] (SMPH). In some such embodiments, the two reaction products thus obtained are mixed together, usually causing a reaction of the thiol groups contained in one batch of modified streptavidin muteins with the activated amino acids (e.g., by maleimide functional groups) of the other batch of modified streptavidin muteins. In some cases, this reaction results in the formation of multimers/oligomers of streptavidin muteins. These oligomers have any suitable number of individual molecules, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more, and the degree of oligomerization can vary depending on the reaction conditions.

In some embodiments, the oligomeric or polymeric reagents (e.g., selection or stimulation reagents) can be isolated by size exclusion chromatography, and any desired fractions can be used as reagents. For example, in some embodiments, after reacting a streptavidin mutein modified in the presence of a heterobifunctional cross-linker such as 2-iminothiolane and sulfo-SMCC, the oligomeric or polymeric reagents can be isolated by size exclusion chromatography, and any desired fractions can be used as reagents. In some embodiments, the oligomers do not (and need not) have a single molecular weight, but the oligomers can observe a statistical weight distribution, such as a Gaussian distribution. In some cases, any oligomer having more than three streptavidin tetramers or mutein tetramers, e.g., homo- or hetero-tetramers, may be used as a soluble reagent, typically 3-50 tetramers, e.g., homo- or hetero-tetramers, 10-40 tetramers, e.g., homo- or hetero-tetramers, or 25-35 tetramers, e.g., homo- or hetero-tetramers. The oligomer may have, e.g., 3-25 streptavidin mutein tetramers, e.g., homo- or hetero-tetramers. In some aspects, the streptavidin mutein has a molecular weight of about 50 kDa and the soluble oligomer is from about 150 kDa to about 2000 kDa, from about 150 kDa to about 1500 kDa, from about 150 kDa to about 1250 kDa, from about 150 kDa to 1000 kDa, from about 150 kDa to about 500 kDa or from about 150 kDa to about 300 kDa, from about 300 kDa to about 2000 kDa, from about 300 kDa to about 1500 kDa, from about 300 kDa to about 1250 kDa, from about 300 kDa to 1000 kDa, from about 300 kDa to about 500 kDa, from about 500 kDa to about 2000 kDa, from about 500 kDa to about 1500 kDa, about 500 kDa to about 1250 kDa, about 500 kDa to 1000 kDa, about 1000 kDa to about 2000 kDa, about 1000 kDa to about 1500 kDa, about 1000 kDa to about 1250 kDa, about 1250 kDa to about 2000 kDa or about 1500 kDa to about 2000 kDa. Typically, each streptavidin molecule/mutein has four biotin binding sites, so that such a reagent may provide 12 to 160 binding sites Z, e.g., 12 to 100 binding sites Z.

B. Agents Agents contemplated herein include selection agents and stimuli. In some embodiments, the selection agent and the stimuli are the same. In some embodiments, the selection agent and the stimuli are different. However, both the selection agent and the stimuli agent may be formulated from the same substance. In some embodiments, the agent (e.g., selection agent or stimuli) has one or more binding sites B for binding to a molecule on a cell surface, such as a cell surface molecule. Thus, in some examples, the agent (e.g., selection agent or stimuli) contains a binding site B or multiple binding sites B, and the specific binding of the agent (e.g., selection agent or stimuli) to a molecule on the surface of a target cell contains an interaction between B and the molecule. In some embodiments, the agent contains only a single binding site, i.e., a monovalent binding site. In some embodiments, the agent (e.g., selection agent or stimuli) has at least two binding sites B, such as multiple binding sites B including three, four, or five binding sites B that have the ability to bind to a cell surface molecule. In some such aspects, at least two or more binding sites B may be identical. In some embodiments, one or more of the at least two or more binding sites B may be different (e.g., B1 and B2).

In some embodiments, one or more different agents (e.g., one or more different, e.g., selection agents or stimuli or other agents that bind to molecules on cells) are reversibly bound to a reagent (e.g., selection agent or stimuli reagent). In some embodiments, at least two, three, four or more different agents (e.g., selection agents or stimuli) are reversibly bound to the same reagent. In some embodiments, at least two different agents (e.g., selection agents or stimuli) are reversibly bound to the same reagent, whereby each agent contains a binding site B or multiple binding sites B for specific binding between the agent and the molecule. In some embodiments, at least two or more agents (e.g., selection agents or stimuli) contain the same binding site B, e.g., for binding to the same molecule or substantially the same molecule. In some embodiments, at least two or more agents (e.g., selection agents or stimuli) contain different binding sites B for binding to different molecules. In some embodiments, the first agent (e.g., the first agent or the first stimulator) contains binding site B1, B2, B3, B4, etc., and the second agent (e.g., the second agent or the second stimulator) contains another of binding sites B1, B2, B3, B4, etc. In some embodiments, the first agent (e.g., the first agent) contains binding site B1, and the second agent (e.g., the second agent) contains binding site B3. In some embodiments, the first agent (e.g., the first stimulator) contains binding site B2, and the second agent (e.g., the second stimulator) contains binding site B4. In any of these embodiments, the first agent and the second agent can contain binding partner C1 or binding partner C2. In some embodiments, C1 and C2 can be the same. In some embodiments, C1 and C2 are different. In some embodiments, the first agent and the second agent contain the same binding partner C1.

In some cases, the dissociation constant (K _D ) for binding between an agent (e.g., via binding site B) and binding site Z of a reagent may have a value ranging from about 10 ⁻² M to about 10 ⁻¹³ M, or from about 10 ⁻³ M to about 10 ⁻¹² M, or from about 10 ⁻⁴ M to about 10 ⁻¹¹ M, or from about 10 ⁻⁵ M to about 10 ⁻¹⁰ M. In some embodiments, the dissociation constant (K _D ) for binding between a binding agent and a molecule is such that it is a low affinity, e.g., with a K _D ranging from about 10 ⁻³ to about 10 ⁻⁷ M. In some embodiments, the dissociation constant (K _D ) for binding between a binding agent and a molecule is such that it is a high affinity, e.g., with a K _D ranging from about 10 ⁻⁷ to about 1×10 ⁻¹⁰ M.

In some embodiments, dissociation of the binding of the agent via binding site B and the molecule occurs sufficiently fast to, for example, stain the target cell only transiently or to allow association with the agent after disruption of the reversible bond between the reagent and the agent. In some cases, when expressed in terms of the k _off rate (also called the dissociation rate constant) for binding between an agent and a molecule (via binding site B), the k _off rate can be at or greater than about 0.5×10 ⁻⁴ sec ⁻¹ , at or greater than about ¹ × ^{10 −4 sec −1} , at or ^greater than about 2× ^{10 −4 sec −1} , at ^or greater than about 3× ^{10 −4} sec ⁻¹ , at or greater than about 4 ^{× 10 −4} sec ⁻¹ , at ^or greater than about 5× ^{10 −4 sec −1} , at or greater ^than about 1× ^{10 −3 sec −1 ,} at ^or greater than about 1.5 ^{× 10 −3 sec -1} or greater, about ^{2x10-3sec - 1} or greater, about 3x10-3sec- ¹ or greater, about ^{4x10-3sec-1 , about 5x10-3sec- 1 or greater ,} about 1x10-2sec ^{or greater} , or about ^{5x10-1sec -} 1 or ^greater . It ^is within the level of one of ordinary skill in the art to empirically determine the k _off rate range suitable for a particular agent and cell-molecule interaction (see ^, for example, US Patent Application Publication No. 2014/0295458). For example, an agent with a somewhat ^high k _off rate, for example, greater than ^{4.0x10-4sec -1,} may be used so that the majority of the agent can be removed or separated within 1 hour after disruption of the binding complex. In other cases, an agent having a k _off rate of, for example, 1.0×10 ⁻⁴ sec ⁻¹ may be used such that following disruption of the binding complex, the majority of the agent can be removed or separated from the cell within about three and a half hours.

In some embodiments, the K _D of this binding, as well as the K _D rate, k off rate and k _on rate of this binding formed between binding site B of an agent (e.g., a selection agent or stimulatory agent) and a cell surface _molecule , may be determined by any suitable means, such as, for example, fluorescence titration, equilibrium dialysis or surface plasmon resonance.

In some aspects, a cell surface molecule is a molecule to which an agent (e.g., a selection agent or a stimulant) can be directed. In some embodiments, the cell surface molecule is a peptide or protein, e.g., a receptor, such as a membrane receptor protein. In some embodiments, the receptor is a lipid, polysaccharide, or nucleic acid. In some embodiments, a cell surface molecule that is a protein can be a peripheral membrane protein or an integral membrane protein. The cell surface molecule can have one or more domains that span the membrane in some embodiments. As a few examples, membrane proteins having a transmembrane domain include olfactory receptors, rhodopsin receptors, rhodopsin pheromone receptors, peptide hormone receptors, taste receptors, G-protein coupled receptors such as GABA receptors, opiate receptors, serotonin receptors, Ca2+ receptors, melanopsin, and other G-protein coupled receptors, acetylcholine receptors, nicotinic receptors, adrenergic receptors, noradrenergic receptors, catecholamine receptors, L-DOPA receptors, and dopamine and serotonin (biogenic amines, endorphins/enkephalins) neuropeptide receptors, as well as neurotransmitter receptors such as ligand-gated, voltage-gated, or mechanically-gated receptors. The receptor may be a receptor for a substance that reaches the target cell, a receptor kinase such as a serine kinase/threonine kinase or a tyrosine kinase, a porin/channel such as a chloride channel, a potassium channel or a sodium channel, an OMP protein, an amino acid transporter, an ABC transporter (ATP-binding cassette transporter) such as a Na-glucose transporter or a Na/iodine transporter, a light-harvesting complex, a cytochrome c oxidase, an ion transporter such as an ATPase Na/K, an ATPase H/K or an ATPase Ca, a metalloprotease, or a cell adhesion receptor such as an integrin or a catherin.

In some embodiments, the cell surface molecule can be an antigen that defines a desired cell population or cell subpopulation, such as a population or subpopulation of blood cells, such as lymphocytes (e.g., T cells, T helper cells, e.g., CD4+ T helper cells, B cells, or natural killer cells), monocytes, or stem cells, such as CD34-positive peripheral stem cells or Nanog- or Oct-4-expressing stem cells. Examples of T cells include CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Tregs). Specific examples of Tregs include CD4 CD25 CD45RA Treg cells, and specific examples of memory T cells include CD62L CD8+ specific central memory T cells. The cell surface molecule can be a marker for tumor cells.

As described above, in some embodiments, the agent (e.g., a selection agent or stimulator) has a binding partner C in addition to a binding site B that is capable of binding to a cell surface molecule. In some aspects, this binding partner C is capable of binding to a binding site Z of a reagent (e.g., a selection agent or stimulator (e.g., an oligomeric stimulatory reagent)), which has one or more binding sites for the binding partner C. In some embodiments, the non-covalent bond that may be formed between the binding partner C contained in the agent (e.g., a selection agent or stimulator) and the binding site(s) Z of the reagent (e.g., a selection agent or stimulator (e.g., an oligomeric stimulatory reagent)) may be of any desired strength and affinity, which may be disruptable or reversible under the conditions in which the method is performed. The agent (e.g., a receptor-binding agent or a selection agent) can include at least one, two, three or more additional binding partners C, and the reagent (e.g., a selection agent or a stimulating agent (e.g., an oligomeric stimulating agent)) can include at least two, e.g., three, four, five, six, seven, eight or more binding sites Z for the binding partner C included in the agent (e.g., the selection agent or the stimulating agent). Any combination of binding partner C and reagent with one or more corresponding binding sites Z can be selected such that, for example, the binding partner C and binding site Z can bind reversibly in a complex, resulting in, for example, an avidity effect, as described in U.S. Pat. Nos. 7,776,562, 8,298,782 or WO 2002/054065.

The binding partner C included in the agent (e.g., the selection agent or stimulant) may be, for example, hydrocarbon-based (including polymeric) and may include nitrogen, phosphorus, sulfur, carbene, halogen or pseudohalogen groups. In some aspects, it may be an alcohol, an organic acid, an inorganic acid, an amine, a phosphine, a thiol, a disulfide, an alkane, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a sugar, an oligosaccharide or a polysaccharide. As further examples, it may be a cation, an anion, a polycation, a polyanion, a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube or a carbon nanofoam. In general, such a binding partner C has a higher affinity for the binding site of the reagent than other entities. Examples of respective binding partners C include, but are not limited to, crown ethers, immunoglobulins, fragments thereof and proteinaceous binding molecules with antibody-like functions.

In some embodiments, the binding partner C in the agent (e.g., the selection agent or stimulant) comprises biotin, and the reagent comprises a streptavidin analog or an avidin analog that reversibly binds to biotin. In some embodiments, the binding partner C in the agent (e.g., the selection agent or stimulant) comprises a biotin analog that reversibly binds to streptavidin or avidin, and the reagent comprises a streptavidin, avidin, streptavidin analog, or avidin analog that reversibly binds to the respective biotin analog. In some embodiments, the binding partner C in the agent (e.g., the selection agent or stimulant) comprises a streptavidin-binding peptide or an avidin-binding peptide, and the reagent comprises a streptavidin, avidin, streptavidin analog, or avidin analog that reversibly binds to the respective streptavidin-binding peptide or avidin-binding peptide.

In some embodiments, the reagent (e.g., the selection or stimulating agent) is streptavidin, such as a streptavidin mutein (e.g., as set forth in SEQ ID NOs:3-6, 104, 105), including any of those described above, and the binding partner C included in the agent (e.g., the selection or stimulating agent) may comprise a streptavidin-binding peptide. In some embodiments, the streptavidin-binding peptide may comprise a sequence of the general formula set forth in SEQ ID NO:9, e.g., contains a sequence set forth in SEQ ID NO:10. In some embodiments, the peptide sequence has the general formula set forth in SEQ ID NO:11, e.g., the general formula set forth in SEQ ID NO:12. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag®, as set forth in SEQ ID NO:7). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II and shown in SEQ ID NO:8). In some embodiments, the peptide ligand contains a continuous sequence of at least two streptavidin binding modules, the distance between the two modules is at least 0 amino acids and less than 50 amino acids, one binding module has 3-8 amino acids and contains at least the sequence His-Pro-Xaa (SEQ ID NO:9), where Xaa is glutamine, asparagine or methionine. Another binding module is the same streptavidin peptide ligand as shown in SEQ ID NO:11 or a different streptavidin peptide ligand (see, e.g., International Published PCT Application WO02/077018; U.S. Patent No. 7,981,632). In some embodiments, the peptide ligand contains a sequence having a formula shown in either SEQ ID NO:13 or 14. In some embodiments, the peptide ligand has a sequence of amino acids set forth in any of SEQ ID NOs:15-19. In many cases, these streptavidin-binding peptides all bind to the same binding site, i.e., the biotin-binding site of streptavidin. When one or more such streptavidin-binding peptides are used as binding partner C, e.g., C1 and C2, the multimerization reagent is typically a streptavidin mutein.

In some embodiments, the streptavidin-binding peptide may be further modified. In some embodiments, the streptavidin-binding peptide may comprise the peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also called Strep-tag® II and shown in SEQ ID NO:8) conjugated to nickel-loaded Tris-NTA (also called His-STREPPER or His/Strep-tag® II adaptor).

In some embodiments, the binding partner C of an agent (e.g., a receptor-binding agent or a selection agent) includes a moiety known to those of skill in the art as an affinity tag. In such embodiments, the reagent can include a corresponding binding partner, e.g., an antibody or antibody fragment, that is known to bind to the affinity tag. As some specific examples of known affinity tags, the binding partner C contained within the agent (e.g., selection agent or stimulatory agent) can be dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, maltose binding protein, glutathione-S-transferase (GST), chitin binding protein (CBP) or thioredoxin, calmodulin binding peptide (CBP), FLAG'-peptide, HA-tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) (SEQ ID NO:20), VSV-G-tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys) (SEQ ID NO:21), HSV-tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp) (SEQ ID NO:22), SEQ ID NO:22), the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly) (SEQ ID NO:23), maltose binding protein (MBP), the HSV epitope of herpes simplex virus glycoprotein D with the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO:24), the "myc" epitope of the transcription factor c-myc with the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO:25), the V5-tag (sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) (SEQ ID NO:26), or glutathione-S-transferase (GST). In such embodiments, the complex formed between one or more binding sites Z of the reagent, which may be an antibody or antibody fragment, and the antigen can be competitively disrupted by adding free antigen, i.e., a free peptide (epitope tag) or a free protein (such as MBP or CBP). In some embodiments, the affinity tag may be an oligonucleotide tag. In some cases, such an oligonucleotide tag may be used, for example, to hybridize to an oligonucleotide with a complementary sequence linked to or contained in the reagent.

Further examples of suitable binding partners C include, but are not limited to, lectins, protein A, protein G, metals, metal ions, nitrilotriacetic acid derivatives (NTA), RGD motifs, dextran, polyethyleneimine (PEI), redox polymers, glycoproteins, aptamers, dyes, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, polyU or oligo-dT. Lectins such as concanavalin A are known to bind polysaccharides or glycosylated proteins. Specific examples of dyes are triazine dyes such as Cibacron Blue F3G-A (CB) or Red HE-3B, which specifically bind NADH-dependent enzymes. Green A typically binds CoA proteins, human serum albumin, and dehydrogenases. In some cases, the dyes 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole bind to DNA. In general, metal cations such as Ni, Cd, Zn, Co, or Cu are commonly used to bind affinity tags such as hexahistidine or oligohistidine-containing sequences, including His-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys tags (MAT tags) (SEQ ID NO:35) and N-methacryloyl-(L)-cysteine methyl ester.

In some embodiments, binding between a binding partner C included in an agent (e.g., a selection agent or a stimulant) and one or more binding sites Z of a reagent occurs in the presence of divalent, trivalent or tetravalent cations. In this regard, in some embodiments, the reagent includes divalent, trivalent or tetravalent cations, typically held in combination, e.g., by the use of a suitable chelator. In some embodiments, the binding partner C included in an agent (e.g., a selection agent or a stimulant) may include, for example, a moiety that includes a complex, a divalent, trivalent or tetravalent cation. Examples of respective metal chelators include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 2,3-dimer-capto-1-propanol (dimercaprol), porphine and heme. As an example, EDTA forms complexes with up to monovalent, up to divalent, up to trivalent and up to tetravalent metal ions, such as silver (Ag ⁺ ), calcium (Ca ²⁺ ), manganese (Mn ^{2+ ), copper (Cu 2+ ), iron (Fe 2+ ) ,} cobalt (Co ⁺ ) and zirconium (Zr ⁴⁺ ), while BAPTA is specific for Ca ²⁺ . As a specific example, a standard method used in the art is to form a complex between an oligohistidine tag and copper (Cu ²⁺ ), nickel (Ni ²⁺ ), cobalt (Co ²⁺ ), or zinc (Zn ²⁺ ) ions, which are presented by the chelating agent nitrilotriacetic acid (NTA).

In some embodiments, the binding partner C contained in the agent (e.g., a selection agent or a stimulant) comprises a calmodulin-binding peptide, and the reagent comprises a multimeric calmodulin, e.g., as described in U.S. Pat. No. 5,985,658. In some embodiments, the binding partner C contained in the agent (e.g., a selection agent or a stimulant) comprises a FLAG peptide, and the reagent comprises an antibody that binds to the FLAG peptide. For example, the FLAG peptide binds to monoclonal antibody 4E11, as described in U.S. Pat. No. 4,851,341. In one embodiment, the binding partner C contained in the agent (e.g., a selection agent or a stimulant) comprises an oligohistidine tag, and the reagent comprises an antibody or a transition metal ion that binds to the oligohistidine tag. In some cases, disruption of all of these binding complexes may be achieved by chelation of the metal ion, e.g., chelation of calcium, such as by adding EDTA or EGTA. In some embodiments, calmodulin, an antibody such as 4E11, or a chelated metal ion or a free chelator may be multimerized by conventional methods. This can be achieved, for example, by biotin labeling or conjugation with streptavidin or avidin or oligomers thereof, or in a first step, by introducing carboxyl residues, e.g. dextran, into the polysaccharide essentially as described in Noguchi, A, et al, Bioconjugate Chemistry (1992) 3, 132-137, and in a second step, by coupling calmodulin or an antibody or a chelated metal ion or a free chelator, via a primary amino group, to the carboxyl groups in the polysaccharide backbone, e.g. dextran, using conventional carbodiimide chemistry. In some such embodiments, the bond between a binding partner C contained in the agent (e.g., a selection agent or a stimulant) and one or more binding sites Z of the reagent can be disrupted by chelation of the metal ion. Metal chelation can be achieved, for example, by the addition of EGTA or EDTA.

In some embodiments, the agent (e.g., selection agent or stimulatory agent) that specifically binds to a cell surface molecule may be comprised, for example, in an antibody, a fragment thereof, or a proteinaceous binding molecule with antibody-like functions. In some embodiments, the binding site B of the agent is a binding site of an antibody, e.g., is or contains one or more complementarity determining regions (CDRs) of an antibody. Examples of (recombinant) antibody fragments include, but are not limited to, Fab fragments, Fv fragments, single chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, diabodies, triabodies (Iliades, P., et al., FEB S Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.J., et al., Trends Biotechnol. (2003), 21, 11, 484-490). In some embodiments, the agent (e.g., receptor binding agent or selection agent) may comprise a bivalent proteinaceous artificial binding molecule, such as a dimeric lipocalin mutein, also known as "duocalin".

In some embodiments, the agent (e.g., selection agent or stimulator) may have a single binding site B, i.e., it may be monovalent. Examples of monovalent agents (e.g., selection agents or stimulators) include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single chain Fv fragments (scFv), including bivalent single chain Fv fragments.

In some embodiments, the agent (e.g., selection agent or stimulator) is an antibody or an antigen-binding fragment of an antibody, such as, for example, a bivalent antibody fragment, such as a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), or an F(ab') ₂ -fragment. In some embodiments, the agent (e.g., selection agent or stimulator) is or is derived from a parent antibody known to bind to a cellular molecule of interest. Various antibody molecules or fragments thereof against cell surface molecules are well known in the art, and any of a variety of such may be used as an agent in the methods herein. In some embodiments, the agent (e.g., selection agent or stimulator) is an antibody or fragment thereof that contains one or more amino acid substitutions in the variable heavy chain of a parent or reference antibody, for example, to generate an antibody that has an altered affinity or exhibits a sufficiently fast off-rate as described above. For example, exemplary of such mutations are known in the context of variants of the anti-CD4 antibody 13B8.2 (see, e.g., U.S. Pat. No. 7,482,000, U.S. Patent Application Publication No. 2014/0295458 or International Patent Application No. WO 2013/124474), any of which may be generated in another parent or reference antibody.

In some aspects, agents that can be monovalent (e.g., selection agents or stimulants) include, for example, monovalent antibody fragments or monovalent artificial binding molecules (proteinaceous or otherwise), such as muteins based on the lipocalin family of polypeptides (also known as "Anticalin" (registered trademark)), or bivalent molecules, such as antibodies or fragments in which both binding sites are retained, such as F(ab') ₂ fragments.

An example of a proteinaceous binding molecule with antibody-like functions includes muteins based on polypeptides of the lipocalin family (see, for example, WO 03/029462; Beste et al, Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903). In general, for example bilin-binding protein, human neutrophil lipocalin gelatinase-associated lipocalin, human Apolipoprotein D or human tear lipocalin have natural ligand binding sites that can be modified so that they bind to a given target. Further examples of proteinaceous binding molecules with antibody-like binding properties that can be used as agents (e.g., selection or stimulating agents) that specifically bind to cell surface molecules include, but are not limited to, the so-called glubodies (see, e.g., International Patent Application No. WO 96/23879), proteins based on ankyrin scaffolds (Mosavi, L.K., et al, Protein Science (2004) 13, 6, 1435-1448) or crystal scaffolds (e.g., International Patent Application No. WO 01/04144), proteins described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, AdNectins, adnectins, tetranectins and avimers. Avimers, including multivalent avimer proteins developed by exon shuffling of a family of human receptor domains, generally contain so-called A domains that arise as strings of domains in multiple cell surface receptors (Silverman, J., et al, Nature Biotechnology (2005) 23, 1556-1561). Adnectins are generally derived from domains of human fibronectin, but generally contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins are generally derived from the respective human homotrimeric protein, but generally also contain loop regions in the C-type lectin domain that can be engineered for desired binding. Peptoids are oligo(N-alkyl)glycines that can act as protein ligands in some cases, but usually differ from peptides in that their side chains are attached to amide nitrogens rather than carbon atoms. Peptoids are generally resistant to proteases and other modifying enzymes and can have much higher cell permeability than peptides (see, e.g., Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509).

Further examples of suitable proteinaceous binding molecules include EGF-like domains, Kringle domains, fibronectin type I domains, fibronectin type II domains, fibronectin type III domains, PAN domains, Gla domains, SRCR domains, Kunitz/bovine pancreatic trypsin inhibitor domains, tendamistat, Kazal-type serine protease inhibitor domains, trefoil (P-type) domains, von Willebrand factor type C domains, anaphylatoxin-like domains, CUB domains, thyroglobulin type I lipids, , LDL receptor class A domain, sushi domain, link domain, thrombospondin type I domain, immunoglobulin domain or immunoglobulin-like domain (e.g. domain antibody or camel heavy chain antibody), C-type lectin domain, MAM domain, von Willebrand factor type A domain, somatomedin B domain, WAP type 4 disulfide core domain, F5/8 type C domain, hemopexin domain, SH2 domain, SH3 domain, laminin type EGF-like domain, C2 domain, "kappa body" (Ill.et al., Protein Eng (1997) 10, 949-57, the so-called "minibodies" (Martin et al., EMBO J (1994) 13, 5303-5309), diabodies (Holliger et al., PNAS USA (1993) 90, 6444-6448), the so-called "Janusis" (Traunecker et al., EMBO J (1991) 10, 3655-3659 or Traunecker et al., Int J Cancer (1992) Suppl 7,51-52), nanobodies, microbodies, affilins, affibodies, knottins, ubiquitins, zinc finger proteins, autofluorescent proteins, or leucine-rich repeat proteins. In some embodiments, an example of a nucleic acid molecule with antibody-like function is an aptamer. Generally, aptamers fold into a defined three-dimensional motif and exhibit high affinity for a given target structure.

III. Serum-Free Media Formulations and Related Components One or more steps of the provided methods can be performed in the presence of serum-free media containing the free form of glutamine (i.e., L-glutamine) in the basal medium. One or more additional supplements can be added, including one or more supplements containing at least one protein, such as a serum replacement protein, or one or more other components that direct the maintenance, growth and/or expansion of cells. In some embodiments, the serum-free media contains a synthetic amino acid (e.g., a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine). In some embodiments, the concentration of the synthetic amino acid (e.g., a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine) is 0.5 mM or about 0.5 mM to 5 mM or about 5 mM (e.g., 2 mM). In some embodiments, the concentration of L-glutamine is 0.5 mM or about 0.5 mM to 5 mM or about 5 mM (e.g., 2 mM). In some embodiments, at least one protein is a human protein or a recombinant protein, such as a serum replacement protein, such as albumin. In some embodiments, the serum-free medium further comprises one or more recombinant cytokines (e.g., IL-2, IL-7, or IL-15). In some embodiments, the serum-free medium does not further comprise one or more recombinant cytokines (e.g., IL-2, IL-7, or IL-15). In some embodiments, the serum-free medium does not comprise phenol red.

In some embodiments, the serum-free medium provided is generated or prepared from a liquid basal medium and one or more supplements.

In some embodiments, the serum-free medium can contain a synthetic amino acid that can be converted to L-glutamine in cell culture, such as a synthetic amino acid that is a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine. In some cases, the synthetic amino acid is provided in a basal medium that is free of L-glutamine and protein. In some embodiments, the concentration of the synthetic amino acid (e.g., a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine) is at or about 0.5 mM to at or about 5 mM (e.g., 2 mM). In some embodiments, at least one protein is a human-derived protein, a recombinant protein, or both. In some embodiments, the basal medium does not contain phenol red.

In some embodiments, among the supplements for preparing serum-free media are supplements that include at least one protein, where the supplement is frozen or frozen after L-glutamine becomes a component thereof, and a free form of glutamine, e.g., L-glutamine. In some embodiments, the concentration of L-glutamine in the supplement is less than 200 mM, e.g., less than 150 mM, 100 mM or less, e.g., 20 mM to 120 mM, or 40 mM to 100 mM, e.g., about 80 mM. In some embodiments, the concentration of L-glutamine after the supplement is combined with the basal medium is about 0.5 mM to about 5 mM (e.g., 2 mM). In some embodiments, the at least one protein is not of non-mammalian origin. In some embodiments, the at least one protein is a human protein or a human-derived protein, or is recombinant. In some embodiments, the at least one protein includes albumin, e.g., human or recombinant human albumin.

A. Basal Medium In some embodiments, the basal medium comprises a carbon source such as glucose, water, one or more salts, as well as a source of amino acids and nitrogen.

In some embodiments, the basal medium comprises amino acids. In some embodiments, the amino acids comprise aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, norvaline, tryptophan, phenylalanine, isoleucine, leucine, lysine, hydroxyproline, sarcosine, and/or proline.

In some embodiments, the basal medium comprises at least one synthetic amino acid. In some embodiments, the synthetic amino acid can be converted to the free form of glutamine (i.e., L-glutamine) in a cell culture comprising the cells. In some embodiments, the cells comprise human cells. In some embodiments, the cells comprise immune cells. In some embodiments, the cells are genetically engineered cells. In some embodiments, the cells are T cells. In some embodiments, the cells are genetically engineered T cells. In some embodiments, the cells are genetically engineered to express a recombinant receptor (e.g., a chimeric antigen receptor). In some embodiments, the cells are chimeric antigen receptor (CAR)-expressing T cells.

In some embodiments, the synthetic amino acid is a stabilized form of glutamine (i.e., L-glutamine). In some embodiments, the synthetic amino acid is more stable than glutamine (i.e., L-glutamine) in an aqueous solution (e.g., basal medium). In some embodiments, the synthetic amino acid does not produce a significant amount of glutamine in the basal medium. In some embodiments, the synthetic amino acid does not produce a significant amount of pyrrolidone carboxylic acid or ammonia in the basal medium. In some embodiments, the synthetic amino acid does not produce a significant amount of glutamine (i.e., L-glutamine) in the basal medium for at least about 1, 3, 5, 7, 9, 11, 13, or 14 days. In some embodiments, the synthetic amino acid does not produce a significant amount of glutamine (i.e., L-glutamine) in the basal medium for at least about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the synthetic amino acids do not produce significant amounts of glutamine (i.e., L-glutamine) in the basal medium for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the synthetic amino acids do not produce significant amounts of glutamine (i.e., L-glutamine) in the basal medium for at least about 1, 2, 3, 4, or 5 years. In some embodiments, the synthetic amino acids do not produce significant amounts of pyrrolidone carboxylic acid or ammonia in the basal medium for at least about 1, 3, 5, 7, 9, 11, 13, or 14 days. In some embodiments, the synthetic amino acids do not produce significant amounts of pyrrolidone carboxylic acid or ammonia in the basal medium for at least about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the synthetic amino acids do not produce significant amounts of pyrrolidone carboxylic acid or ammonia in the basal medium for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the synthetic amino acid does not produce significant amounts of pyrrolidone carboxylic acid or ammonia in the basal medium for at least 1, 2, 3, 4, or 5 years.

In some embodiments, the synthetic amino acids are soluble in an aqueous solution (e.g., a basal medium). In some embodiments, the solubility of the synthetic amino acids in an aqueous solution is greater than the free form of glutamine (i.e., L-glutamine).

In some embodiments, the synthetic amino acid can be delivered to a cell where it can be converted to the free form of glutamine (i.e., L-glutamine). In some embodiments, the cell comprises an immune cell. In some embodiments, the cell is a genetically engineered cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a genetically engineered T cell. In some embodiments, the cell is genetically engineered to express a recombinant receptor (e.g., a chimeric antigen receptor). In some embodiments, the cell is a chimeric antigen receptor (CAR)-expressing T cell.

In some embodiments, the synthetic amino acid is a dipeptide. In some embodiments, the synthetic amino acid is a tripeptide. In some embodiments, the synthetic amino acid is a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™, which does not spontaneously degrade in basal medium.

In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the basal medium is about 0.5 mM to 5 mM. In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the basal medium is at or about 2 mM. In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) is between 0.5 mM and 1 mM, 0.5 mM and 1.5 mM, 0.5 mM and 2 mM, 0.5 mM and 2.5 mM, 0.5 mM and 3 mM, 0.5 mM and 3.5 mM, 0.5 mM and 4 mM, 0.5 mM and 4.5 mM, 0.5 mM and 5 mM, 1 mM and 1.5 mM, 1 mM and 2 mM, 1 mM and 2.5 mM, 1 mM and 3 mM, 1 mM and 3.5 mM, 1 mM and 4 mM, 1 mM and 4.5 mM, 1 mM and 5 mM, 1.5 mM and 2 mM, 1.5 mM and 2.5 mM, 1.5 mM and 3 mM, 1.5 mM and 3.5 mM, 1.5 mM and 4 mM, 1.5 mM and 4.5 mM, 1.5 mM and 5 mM, 2 mM and 2.5 mM, 2 mM and 3 mM, 2 0.5 mM to 3.5 mM, 2 mM to 4 mM, 2 mM to 4.5 mM, 2 mM to 5 mM, 2.5 mM to 3 mM, 2.5 mM to 3.5 mM, 2.5 mM to 4 mM, 2.5 mM to 4.5 mM, 2.5 mM to 5 mM, 3 mM to 3.5 mM, 3 mM to 4 mM, 3 mM to 4.5 mM, 3 mM to 5 mM, 3.5 mM to 4 mM, 3.5 mM to 4.5 mM, 3.5 mM to 5 mM, 4 mM to 4.5 mM, 4 mM to 5 mM or 4.5 mM to 5 mM, or about 0.5 mM to 1 mM, about 0.5 mM to 1.5 mM, about 0.5 mM to 2 mM, about 0.5 mM to 2.5 mM, about 0.5 mM to 3 mM, about 0.5 mM to 3.5 mM, about 0.5 mM to 4 mM, about 0.5 mM to 4.5 mM, about 0.5 mM to 5 mM, about 1 mM to 1.5 mM, about 1 mM to 2 mM, about 1 mM to 2.5 mM, about 1 mM to 3 mM, about 1 mM to 3.5 mM, about 1 mM to 4 mM, about 1 mM to 4.5 mM, about 1 mM to 5 mM, about 1.5 mM to 2 mM, about 1.5 mM to 2.5 mM, about 1.5 mM to 3 mM, about 1.5 mM to 3.5 mM, about 1.5 mM to 4 mM, about 1.5 mM to 4.5 mM, about 1.5 mM to 5 mM, about 2 mM to 2.5 mM, about 2 mM to 3 mM, about 2 mM to 3.5 mM, about 2 mM to 4 mM, about 2 mM to 4.5 mM, about 2 mM to 5 mM, about 2.5 mM to 3 mM, about 2.5 mM to 3.5 mM, about 2.5 mM to 4 mM, about 2.5 mM to 4.5 mM, about 2.5 mM to 5 mM, about 3 mM to 3.5 mM, about 3 mM to 4 mM, about 3 mM to 4.5 mM, about 3 mM to 5 mM, about 3.5 mM to 4 mM, about 3.5 mM to 4.5 mM, about 3.5 mM to 5 mM, about 4 mM to 4.5 mM, about 4 mM to 5 mM or about 4.5 mM to 5 mM (each of the end values being inclusive). In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the basal medium is between 5 mM and 7.5 mM, between 5 mM and 10 mM, between 5 mM and 12.5 mM, between 5 mM and 15 mM, between 5 mM and 17.5 mM, between 5 mM and 20 mM, between 7.5 mM and 10 mM, between 7.5 mM and 12.5 mM, between 7.5 mM and 15 mM, between 7.5 mM and 17.5 mM, between 7.5 mM and 20 mM, between 10 mM and 12.5 mM, between 10 mM and 15 mM, between 10 mM and 17.5 mM, between 10 mM and 20 mM, between 12.5 mM and 15 mM, between 12.5 mM and 17.5 mM, between 12.5 mM and 20 mM, between 15 mM and 17.5 mM, between 15 mM and 20 mM, or between 17.5 mM and 20 mM, or between about 5 mM and 7.5 mM, between about 5 mM and 10 mM, between about about 5 mM to 12.5 mM, about 5 mM to 15 mM, about 5 mM to 17.5 mM, about 5 mM to 20 mM, about 7.5 mM to 10 mM, about 7.5 mM to 12.5 mM, about 7.5 mM to 15 mM, about 7.5 mM to 17.5 mM, about 7.5 mM to 20 mM, about 10 mM to 12.5 mM, about 10 mM to 15 mM, about 10 mM to 17.5 mM, about 10 mM to 20 mM, about 12.5 mM to 15 mM, about 12.5 mM to 17.5 mM, about 12.5 mM to 20 mM, about 15 mM to 17.5 mM, about 15 mM to 20 mM or about 17.5 mM to 20 mM (each of which includes both end values). In some embodiments, the concentration of dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in basal medium is at least 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM, or at least about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM. In some embodiments, the concentration of dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in basal medium is 2 mM or about 2 mM. In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the basal medium is at most 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM, or 20 mM, or about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 17.5 mM, or about 20 mM.

In some embodiments, the basal medium does not contain L-glutamine or does not contain a significant amount of L-glutamine.

In some embodiments, the basal medium comprises L-glutamine. In some embodiments, the concentration of L-glutamine in the basal medium is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM, or is about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM or about 0.5 mM, or is less than 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM, or is less than about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM. In some embodiments, the concentration of L-glutamine in the basal medium is 1 mM, 2 mM, 3 mM, 4 mM or 5 mM, or is about 1 mM, about 2 mM, about 3 mM, about 4 mM or about 5 mM, or is less than 1 mM, 2 mM, 3 mM, 4 mM or 5 mM, or is less than about 1 mM, 2 mM, 3 mM, 4 mM or 5 mM. In some embodiments, the concentration of L-glutamine in the basal medium is 2 mM or about 2 mM.

In some embodiments, one or more amino acids, such as at least one synthetic amino acid that can be converted to the free form of glutamine (i.e., L-glutamine), such as a dipeptide form of L-glutamine, such as L-alanyl-L-glutamine, are provided in the basal medium. In some embodiments, the basal medium is an artificial or synthetic medium. In some embodiments, the basal medium is or includes a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS). In some embodiments, the basal medium is selected from Dulbecco's modified Eagle's medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), Alpha Minimum Essential Medium (Alpha MEM), Iscove's modified Dulbecco's medium, and M199. In some embodiments, the basal medium is a complex medium (e.g., RPMI-1640, IMDM). In some embodiments, the basal medium is OpTmizer™ CTS™ T Cell Expansion Basal Medium (ThermoFisher).

In some embodiments, the basal medium comprises a nutrient mixture of inorganic salts, sugars, amino acids, and may optionally contain vitamins, organic acids, antioxidants, and/or buffers.

In some embodiments, the basal medium comprises CO ₃ ^2- and HCO ₃ ^- . In some embodiments, the CO ₃ ^2- /HCO ₃ ^- content of the basal medium is balanced with gaseous CO ₂ (e.g., 5-10%), thereby maintaining an optimal pH in the medium. In some embodiments, the basal medium comprises a zwitterion, e.g., HEPES. In some embodiments, the basal medium comprises phenol red. In some embodiments, the basal medium does not comprise phenol red.

In some embodiments, the basal medium includes inorganic salts. In some embodiments, the inorganic salts promote osmotic balance. In some embodiments, the inorganic salts regulate the membrane potential by providing sodium, potassium, and calcium ions.

In some embodiments, the basal medium comprises one or more carbohydrates. In some embodiments, the carbohydrate comprises glucose. In some embodiments, the carbohydrate comprises galactose. In some embodiments, the carbohydrate comprises maltose. In some embodiments, the carbohydrate comprises fructose.

In some embodiments, the basal medium comprises a fatty acid. In some embodiments, the basal medium comprises a lipid. In some embodiments, the basal medium comprises a vitamin (e.g., vitamin A, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin E). In some embodiments, the basal medium comprises a trace element. In some embodiments, the trace element comprises copper. In some embodiments, the trace element comprises zinc. In some embodiments, the trace element comprises selenium. In some embodiments, the trace element comprises a tricarboxylic acid intermediate.

In some embodiments, the basal medium contains a mixture of inorganic salts, sugars, amino acids, and optionally vitamins, organic acids, and/or buffers or other well-known cell culture nutrients. In addition to nutrients, the medium also helps maintain pH and osmolality. In some aspects, the reagents of the basal medium support the growth, proliferation, and/or expansion of cells. A wide variety of basal media are available, including Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Iscove's Modified Dulbecco's Medium, and Ham's Medium. In some embodiments, the basal medium is Iscove's Modified Dulbecco's Medium, RPMI-1640, or α-MEM.

In some embodiments, the basal medium is protein-free. In some embodiments, the basal medium is free of human proteins (e.g., human serum proteins). In some embodiments, the basal medium is serum-free. In some embodiments, the basal medium is free of serum from humans. In some embodiments, the basal medium is free of recombinant proteins. In some embodiments, the basal medium is free of human proteins and recombinant proteins.

In some embodiments, the basal medium comprises a protein or peptide. In some embodiments, the protein is albumin or an albumin substitute. In some embodiments, the albumin is human-derived albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is native human serum albumin. In some embodiments, the albumin is recombinant human serum albumin. In some embodiments, the albumin is recombinant albumin from a non-human source. The albumin substitute can be any protein or polypeptide source. Examples of such protein or polypeptide samples include, but are not limited to, bovine pituitary extract, plant hydrolysate (e.g., rice hydrolysate), bovine fetal albumin (fetuin), egg albumin, human serum albumin (HSA), or another animal-derived albumin, chicken extract, bovine embryo extract, AlbuMAX® I, and AlbuMAX® II. In some embodiments, the protein or peptide comprises transferrin. In some embodiments, the protein or peptide comprises fibronectin. In some embodiments, the protein or peptide comprises aprotinin. In some embodiments, the protein comprises fetuin.

In some embodiments, the basal medium (e.g., OpTmizer™ CTS™ T Cell Expansion Basal Medium) is a liquid formulation. In some embodiments, the basal medium is not frozen or is instructed not to be frozen (e.g., according to its protocol) prior to intended use. In some embodiments, the basal medium is stored at or about 2°C to 8°C. In some embodiments, the basal medium is stored at room temperature. In some embodiments, the basal medium is stable for at least or about 1, 2, 3, 4, 5, or 6 weeks when stored at 2°C to 8°C. In some embodiments, the basal medium is stable for at least or about 1, 2, 3, 4, 5, or 6 months when stored at 2°C to 8°C. In some embodiments, the basal medium is stable for at least or about 1, 2, 3, 4, 5, or 6 months when stored at 2°C to 8°C.

B. Additional Components and Supplements In some embodiments, the serum-free medium comprises a basal medium and one or more additional components that can be provided by one or more supplements. In some embodiments, the one or more supplements comprise at least a first supplement comprising a free form of glutamine (i.e., L-glutamine). In some embodiments, such supplements are frozen prior to use and/or incorporation into the basal medium. In some embodiments, supplements such as those described herein are intended to be used as medium supplements (e.g., medium supplements for basal medium). In some embodiments, the first supplement and/or additional supplements provide cell maintenance. In some embodiments, the cells are primary cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are T cells. In some embodiments, the cells are CD3 T cells, CD4 T cells or CD8 T cells. In some embodiments, the cells are cells of human origin. In some embodiments, the cells are immune cells of human origin. In some embodiments, the cells are T cells of human origin. In some embodiments, the cells are primary immune cells of human origin. In some embodiments, the cells are genetically engineered cells. In some embodiments, the cell is a genetically engineered cell derived from a human. In some embodiments, the cell is a genetically engineered T cell derived from a human (e.g., a chimeric antigen receptor (CAR)-expressing T cell).

In some embodiments, the first supplement is stored or is recommended to be stored at -20°C to 0°C or about -20°C to about 0°C prior to its intended use. In some embodiments, the supplement is stored or is recommended to be stored below about 0°C. In some embodiments, the supplement is frozen immediately or shortly after the free form of glutamine (i.e., L-glutamine) becomes its component until the time the supplement is used for its intended use. In some embodiments, the supplement is frozen the majority of the time after the free form of glutamine (i.e., L-glutamine) becomes its component until the time the supplement is used for its intended use. In some embodiments, the supplement is not kept as a liquid for more than 1, 2, 3, 4, 5, 6, or 7 days after the free form of glutamine (i.e., L-glutamine) becomes its component until the time the supplement is used for its intended use. In some embodiments, the supplement is not kept as a liquid for more than or about 4, 8, 12, 16, 20 or 24 hours after the free form of glutamine (i.e., L-glutamine) becomes a component of the supplement until the time the supplement is used for its intended use. In some embodiments, the supplement is frozen for the majority of the time both before and after the free form of glutamine (i.e., L-glutamine) becomes a component of the supplement until the time the supplement is used for its intended use. In some embodiments, the supplement is at or below room temperature when the free form of glutamine (i.e., L-glutamine) becomes a component of the supplement (e.g., the temperature of the supplement is below or below about 20°C, 15°C, 10°C, 5°C or 0°C). In one aspect, to minimize variable glutamine concentrations and/or ever-increasing ammonia concentrations in serum-free media formulations that may occur due to L-glutamine instability, the presence of L-glutamine in the frozen supplement ensured its stability prior to addition to the basal medium.

In some embodiments, the free form L-glutamine in the supplement does not precipitate when the supplement is thawed. In some embodiments, the free form L-glutamine in the supplement does not precipitate when the supplement is a liquid. In some embodiments, the free form L-glutamine in the supplement does not precipitate when the supplement is thawed at room temperature. In some embodiments, the concentration of the free form of L-glutamine in the supplement is, or is about, 400 mM, 300 mM, 200 mM, 180 mM, 160 mM, 140 mM, 120 mM, 100 mM, or 80 mM, or is less than, or is about, 400 mM, 300 mM, 200 mM, 180 mM, 160 mM, 140 mM, 120 mM, 100 mM, or 80 mM. In some embodiments, the concentration of L-glutamine in the supplement is about 200 mM.

In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the supplement is such that after the supplement is combined with a basal medium (such as that described herein), the concentration of the free form of glutamine (i.e., L-glutamine) in the medium is 0.5 mM to 5 mM or about 0.5 mM to 5 mM. In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the basal medium is 2 mM or about 2 mM. In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) is between 0.5 mM and 1 mM, 0.5 mM and 1.5 mM, 0.5 mM and 2 mM, 0.5 mM and 2.5 mM, 0.5 mM and 3 mM, 0.5 mM and 3.5 mM, 0.5 mM and 4 mM, 0.5 mM and 4.5 mM, 0.5 mM and 5 mM, 1 mM and 1.5 mM, 1 mM and 2 mM, 1 mM and 2.5 mM, 1 mM and 3 mM, 1 mM and 3.5 mM, 1 mM and 4 mM, 1 mM and 4.5 mM, 1 mM and 5 mM, 1.5 mM and 2 mM, 1.5 mM and 2.5 mM, 1.5 mM and 3 mM, 1.5 mM and 3.5 mM, 1.5 mM and 4 mM, 1.5 mM and 4.5 mM, 1.5 mM and 5 mM, 2 mM and 2.5 mM, 2 mM and 3 mM, 2 mM and 3.5 mM, 2 mM and 4 0.5 mM to 1 mM, 2 mM to 4.5 mM, 2 mM to 5 mM, 2.5 mM to 3 mM, 2.5 mM to 3.5 mM, 2.5 mM to 4 mM, 2.5 mM to 4.5 mM, 2.5 mM to 5 mM, 3 mM to 3.5 mM, 3 mM to 4 mM, 3 mM to 4.5 mM, 3 mM to 5 mM, 3.5 mM to 4 mM, 3.5 mM to 4.5 mM, 3.5 mM to 5 mM, 4 mM to 4.5 mM, 4 mM to 5 mM or 4.5 mM to 5 mM, or about 0.5 mM to 1 mM, about 0.5 mM to 1.5 mM, about 0.5 mM to 2 mM, about 0.5 mM to 2.5 mM, about 0.5 mM to 3 mM, about 0.5 mM to 3.5 mM, about 0.5 mM to 4 mM, about 0.5 mM to 4.5 mM, about 0.5 mM to 5 mM, about 1 mM to 1.5 mM, about 1 mM to 2 mM, about 1 about 1 mM to 2.5 mM, about 1 mM to 3 mM, about 1 mM to 3.5 mM, about 1 mM to 4 mM, about 1 mM to 4.5 mM, about 1 mM to 5 mM, about 1.5 mM to 2 mM, about 1.5 mM to 2.5 mM, about 1.5 mM to 3 mM, about 1.5 mM to 3.5 mM, about 1.5 mM to 4 mM, about 1.5 mM to 4.5 mM, about 1.5 mM to 5 mM, about 2 mM to 2.5 mM, about 2 mM to 3 mM, about 2 mM to 3.5 mM, about 2 mM to 4 mM, about 2 mM to 4.5 mM, about 2 mM to 5 mM, about 2.5 mM to 3 mM, about 2.5 mM to 3.5 mM, about 2.5 mM to 4 mM, about 2.5 mM to 4.5 mM, about 2.5 mM to 5 mM, about 3 mM to 3.5 mM, about 3 mM to 4 mM, about 3 mM to 4.5 mM, about 3 mM to 5 mM, about 3.5 mM to 4 mM, about 3.5 mM to 4.5 mM, about 3.5 mM to 5 mM, about 4 mM to 4.5 mM, about 4 mM to 5 mM or about 4.5 mM to 5 mM (each of the end values being inclusive). In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the basal medium is between 5 mM and 7.5 mM, between 5 mM and 10 mM, between 5 mM and 12.5 mM, between 5 mM and 15 mM, between 5 mM and 17.5 mM, between 5 mM and 20 mM, between 7.5 mM and 10 mM, between 7.5 mM and 12.5 mM, between 7.5 mM and 15 mM, between 7.5 mM and 17.5 mM, between 7.5 mM and 20 mM, between 10 mM and 12.5 mM, between 10 mM and 15 mM, between 10 mM and 17.5 mM, between 10 mM and 20 mM, between 12.5 mM and 15 mM, between 12.5 mM and 17.5 mM, between 12.5 mM and 20 mM, between 15 mM and 17.5 mM, between 15 mM and 20 mM, or between 17.5 mM and 20 mM, or between about 5 mM and 7.5 mM, about 5 mM and 10 mM, about 5 mM and 12.5 mM, about 5 mM to 15 mM, about 5 mM to 17.5 mM, about 5 mM to 20 mM, about 7.5 mM to 10 mM, about 7.5 mM to 12.5 mM, about 7.5 mM to 15 mM, about 7.5 mM to 17.5 mM, about 7.5 mM to 20 mM, about 10 mM to 12.5 mM, about 10 mM to 15 mM, about 10 mM to 17.5 mM, about 10 mM to 20 mM, about 12.5 mM to 15 mM, about 12.5 mM to 17.5 mM, about 12.5 mM to 20 mM, about 15 mM to 17.5 mM, about 15 mM to 20 mM or about 17.5 mM to 20 mM (each of which includes both end values). In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the basal medium is at least 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or at least about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM. In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the basal medium is at most 2 mM or about 2 mM.

In some embodiments, the first supplement contains one or more additional components. In some embodiments, an additional supplement, such as a second supplement, is provided to provide one or more additional components. In some embodiments, the supplements, i.e., the first supplement and optionally one or more additional supplements, e.g., the second supplement, are combined with the basal medium to provide the basal medium with one or more additional components.

In some embodiments, the one or more additional components include at least one protein. In some embodiments, the at least one protein is not of non-mammalian origin. In some embodiments, the at least one protein is human or derived from a human. In some embodiments, the at least one protein is recombinant. In some embodiments, the at least one protein includes albumin, transferrin, insulin, fibronectin, aprotinin, or fetuin. In some embodiments, the protein includes one or more of albumin, insulin, or transferrin, optionally one or more of human or recombinant albumin, insulin, or transferrin.

In some embodiments, the protein is albumin or an albumin substitute. In some embodiments, the albumin is a human-derived albumin. In some embodiments, the albumin is a recombinant albumin. In some embodiments, the albumin is a native human serum albumin. In some embodiments, the albumin is a recombinant human serum albumin. In some embodiments, the albumin is a recombinant albumin from a non-human source. The albumin substitute can be any protein or polypeptide source. Examples of such protein or polypeptide samples include, but are not limited to, bovine pituitary extract, plant hydrolysate (e.g., rice hydrolysate), bovine fetal albumin (fetuin), egg albumin, human serum albumin (HSA), or another animal-derived albumin, chicken extract, bovine embryo extract, AlbuMAX® I, and AlbuMAX® II.

In some embodiments, the one or more additional components comprise albumin. In some embodiments, the albumin is human albumin or is derived from human albumin. In some embodiments, the albumin is derived from human serum or human plasma. In some embodiments, the albumin is recombinant albumin. In some embodiments, the recombinant albumin is derived from a human. In some embodiments, the recombinant albumin is not derived from a human. In some embodiments, the supplement comprises natural albumin. In some embodiments, the natural albumin is derived from a human. In some embodiments, the natural albumin is not derived from a human. In some embodiments, the concentration of albumin in the supplement is such that after the supplement is combined with a basal medium (such as those described herein), the concentration or approximate concentration of albumin in the medium is from 0 mg/mL or about 0 mg/mL, 2 mg/mL or about 2 mg/mL, 0 mg/mL or about 0 mg/mL, 4 mg/mL or about 4 mg/mL, 0 mg/mL or about 0 mg/mL, 6 mg/mL or about 6 mg/mL, 0 mg/mL or about 0 mg/mL, 8 mg/mL or about 8 mg/mL, 0 mg/mL or about 0 mg/mL, 10 mg/mL or about 10 mg/mL, 0 mg/mL or about 0 mg/mL, 12 mg/mL or about 12 mg/mL, 2 mg/mL or about 2 mg/mL, 4 mg/mL or about 4 mg/mL, 2 mg/mL or about 2 mg/mL, 6 mg/mL or about 6 mg/mL, 2 mg/mL or about 2 mg/mL, 8 mg/mL or about 8 mg/mL, 10 mg/mL or about 10 mg/mL, 2 mg/mL or about 2 mg/mL to 12 mg/mL or about 12 mg/mL, 4 mg/mL or about 4 mg/mL to 6 mg/mL or about 6 mg/mL, 4 mg/mL or about 4 mg/mL to 8 mg/mL or about 8 mg/mL, 4 mg/mL or about 4 mg/mL to 10 mg/mL or about 10 mg/mL, 4 mg/mL or about 4 mg/mL to 12 mg/mL or about 12 mg/mL, 6 mg/mL or about 6 mg/mL to 8 mg/mL, 6 mg/mL or about 6 mg/mL to 10 mg/mL or about 10 mg/mL, 6 mg/mL or about 6 mg/mL to 12 mg/mL or about 12 mg/mL, 8 mg/mL or about 8 mg/mL to 10 mg/mL In some embodiments, the albumin in the medium is 5 mg/mL or about 5 mg/mL. In some embodiments, the albumin in the medium is 5 mg/mL or about 5 mg/mL.

In some embodiments, the one or more additional components include transferrin or a transferrin substitute. In some embodiments, the transferrin substitute is a compound that can replace transferrin in the supplement to provide substantially similar results to transferrin. Examples of transferrin substitutes include, but are not limited to, any iron chelating compound. Iron chelating compounds that can be used include, but are not limited to, the iron chelates of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), deferoxamine mesylate, dimercaptopropanol, diethylenetriaminepentaacetic acid (DPTA), and trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), as well as ferric citrate chelate and ferrous sulfate chelate. In some embodiments, the transferrin is iron-saturated transferrin. In some embodiments, the transferrin is iron-saturated human transferrin.

In some embodiments, the transferrin or transferrin substitute is human transferrin or is derived from human transferrin. In some embodiments, the transferrin or transferrin substitute is derived from human serum or plasma. In some embodiments, the transferrin or transferrin substitute is recombinant transferrin. In some embodiments, the concentration of transferrin is such that after the supplement is combined with the basal medium (such as those described herein), the concentration of transferrin in the medium is from 10 mg/L or about 10 mg/L, 50 mg/L or about 50 mg/L, 10 mg/L or about 10 mg/L, 100 mg/L or about 100 mg/L, 10 mg/L or about 10 mg/L, 150 mg/L or about 150 mg/L, 10 mg/L or about 10 mg/L, 200 mg/L or about 200 mg/L, 10 mg/L or about 10 mg/L, 250 mg/L or about 250 mg/L, 10 mg/L or about 10 mg/L, 300 mg/L or about 300 mg/L, 10 mg/L or about 10 mg/L, 350 mg/L or about 350 mg/L, 10 mg/L or about 10 mg/L, from 10 mg/L or about 10 mg/L to 400 mg/L or about 400 mg/L, from 10 mg/L or about 10 mg/L to 450 mg/L or about 450 mg/L, from 10 mg/L or about 10 mg/L to 500 mg/L or about 500 mg/L, from 10 mg/L or about 10 mg/L to 550 mg/L or about 550 mg/L, from 10 mg/L or about 10 mg/L to 600 mg/L or about 600 mg/L, from 10 mg/L or about 10 mg/L to 650 mg/L or about 650 mg/L, or from 10 mg/L or about 10 mg/L to 750 mg/L or about 750 mg/L. In some embodiments, the concentration of transferrin is such that after the supplement is combined with a basal medium (such as those described herein), the concentration of transferrin in the medium is at or about 100 mg/L. In some embodiments, the concentration of transferrin is such that after the supplement is combined with a basal medium (such as those described herein), the concentration of transferrin in the medium is at or about 50 mg/L to at or about 150 mg/L.

In some embodiments, the one or more additional components include insulin or an insulin substitute. In some embodiments, the insulin substitute is a zinc-containing compound that can be used in place of insulin to provide substantially similar results as insulin. Examples of insulin substitutes include, but are not limited to, zinc chloride, zinc nitrate, zinc bromide, and zinc sulfate. Several insulins are known to those of skill in the art. See Gilman, A.G. et al, Eds., The Pharmacological Basis of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495. In some embodiments, insulin is used in the supplement and medium rather than an insulin substitute. In some embodiments, the insulin is zinc insulin. In some embodiments, the insulin is human zinc insulin.

In some embodiments, the insulin is human insulin or is derived from human insulin. In some embodiments, the insulin is recombinant insulin. In some embodiments, the insulin is recombinant human insulin. In some embodiments, the concentration of insulin (or insulin substitute) is such that after the supplement is combined with the basal medium (such as those described herein), the approximate concentration of insulin (or insulin substitute) in the medium is from about 1 mg/L to 2.5 mg/L or about 2.5 mg/L, 1 mg/L to 5 mg/L, 1 mg/L to 7.5 mg/L or about 7.5 mg/L, 1 mg/L to 10 mg/L, 1 mg/L to 12.5 mg/L, 1 mg/L to 15 mg/L, 1 mg/L to 17.5 mg/L, 1 mg/L to 20 mg/L, 20 mg/L, 1 mg/L or about 1 mg/L to 22.5 mg/L, 1 mg/L or about 1 mg/L to 25 mg/L, 1 mg/L or about 1 mg/L to 27.5 mg/L, 1 mg/L or about 1 mg/L to 30 mg/L. In some embodiments, the concentration of insulin or insulin substitute in the medium is 10 mg/L or about 10 mg/L. In some embodiments, the concentration of insulin or insulin substitute in the medium is 7.5 mg/L or about 7.5 mg/L to 12.5 mg/L or about 12.5 mg/L.

In some embodiments, the one or more additional components comprise a growth factor. In some embodiments, the growth factor comprises epidermal growth factor (EGF). In some embodiments, the growth factor comprises fibroblast growth factor (FGF). In some embodiments, the growth factor comprises insulin-like growth factor (IGF). In some embodiments, the growth factor comprises nerve growth factor (NGF). In some embodiments, the growth factor comprises platelet-derived growth factor (PDGF). In some embodiments, the growth factor comprises transforming growth factor (TGF).

In some embodiments, the one or more additional components include a hormone (e.g., growth hormone, insulin, hydrocortisone, triiodothyronine, estrogen, androgen, progesterone, prolactin, follicle-stimulating hormone, gastrin-releasing peptide). In some embodiments, the one or more additional components include an alpha globulin or a beta globulin. In some embodiments, the one or more additional components include a peptide or peptide fraction (e.g., a protein hydrolysate derived from an animal, a microorganism, or a plant).

In some embodiments, the one or more additional components comprise a lipid. In some embodiments, the lipid comprises cholesterol. In some embodiments, the lipid comprises a steroid. In some embodiments, the lipid comprises a fatty acid (e.g., palmitic acid, stearic acid, oleic acid, linoleic acid). In some embodiments, the lipid comprises ethanolamine. In some embodiments, the lipid comprises choline. In some embodiments, the lipid comprises inositol.

In some embodiments, the one or more additional components comprise a transition metal. In some embodiments, the transition metal comprises iron. In some embodiments, the transition metal comprises zinc. In some embodiments, the transition metal comprises copper. In some embodiments, the transition metal comprises chromium. In some embodiments, the transition metal comprises iodine. In some embodiments, the transition metal comprises cobalt. In some embodiments, the transition metal comprises selenium. In some embodiments, the transition metal comprises magnesium. In some embodiments, the transition metal comprises molybdenum.

In some embodiments, the one or more additional components comprise vitamins. In some embodiments, the vitamins comprise fat-soluble vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K). In some embodiments, the vitamins comprise water-soluble vitamins (e.g., B1, B2, B6, _B12 , C, folic acid).

In some embodiments, the one or more additional components comprise a polyamine. In some embodiments, the polyamine comprises putrescine. In some embodiments, the polyamine comprises spermidine. In some embodiments, the polyamine comprises spermine.

In some embodiments, the one or more additional components include a reducing agent. In some embodiments, the reducing agent includes 2-mercaptoethanol. In some embodiments, the reducing agent includes alpha-thioglycerol. In some embodiments, the reducing agent includes reduced glutathione.

In some embodiments, the one or more additional components comprise a protective additive. In some embodiments, the protective additive comprises carboxymethylcellulose. In some embodiments, the protective additive comprises polyvinylpyrrolidone. In some embodiments, the protective additive comprises Pluronic F-68. In some embodiments, the protective additive comprises Tween 80.

In some embodiments, the one or more additional components comprise an attachment factor. In some embodiments, the attachment factor comprises fibronectin. In some embodiments, the attachment factor comprises laminin.

In some embodiments, the one or more additional components are one or more of one or more antioxidants, one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, and one or more glucocorticoids. In some embodiments, the antioxidant comprises N-acetyl-L-cysteine, 2-mercaptoethanol or D,L-tocopherol acetate, or derivatives or mixtures thereof. In some embodiments, the albumin is human serum albumin. In some embodiments, the lipid agent comprises Human Ex-Cite® or ethanolamine, or derivatives and mixtures thereof. In some embodiments, the insulin is human zinc insulin. In some embodiments, the transferrin is human iron-saturated transferrin. In some embodiments, the trace element is Se4+. In some embodiments, the glucocorticoid is hydrocortisone. In some embodiments, the supplement is concentrated.

In some embodiments, the one or more additional components include one or more antioxidants, and one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, and one or more glucocorticoids.

In some embodiments, the one or more additional components include one or more of N-acetyl-L-cysteine, human serum albumin, Human Ex-Cyte®, ethanolamine, human zinc insulin, human iron-saturated transferrin, Se4+, hydrocortisone, D,L-tocopherol acetate, and/or 2-mercaptoethanol.

In some embodiments, the one or more additional components include N-acetyl-L-cysteine (NAC). In some embodiments, the concentration of NAC is from 10 mg/L or about 10 mg/L, 50 mg/L or about 50 mg/L, 10 mg/L or about 10 mg/L, 100 mg/L or about 100 mg/L, 10 mg/L or about 10 mg/L, 150 mg/L or about 150 mg/L, 10 mg/L or about 10 mg/L, 200 mg/L or about 200 mg/L, 10 mg/L or about 10 mg/L, 250 mg/L or about 250 mg/L, 10 mg/L or about 10 mg/L, 300 mg/L or about 300 mg/L, 10 mg/L or about 10 mg/L, 350 mg/L or about 350 mg/L, 10 mg/L or about 10 mg/L, mg/L to 400 mg/L or about 400 mg/L, 10 mg/L or about 10 mg/L to 450 mg/L or about 450 mg/L, 10 mg/L or about 10 mg/L to 500 mg/L or about 500 mg/L, 10 mg/L or about 10 mg/L to 550 mg/L or about 550 mg/L, 10 mg/L or about 10 mg/L to 600 mg/L or about 600 mg/L, 10 mg/L or about 10 mg/L to 650 mg/L or about 650 mg/L, 10 mg/L or about 10 mg/L to 700 mg/L or about 700 mg/L.

In some embodiments, the concentration of NAC in the basal medium is from 0 mM or about 0 mM to 1 mM or about 1 mM, from 0 mM or about 0 mM to 2 mM or about 2 mM, from 0 mM or about 0 mM to 3 mM or about 3 mM, from 0 mM or about 0 mM to 4 mM or about 4 mM, from 0 mM or about 0 mM to 5 mM or about 5 mM, from 0 mM or about 0 mM to 6 mM or about 6 mM, from 0 mM or about 0 mM to 7 mM or about 7 mM, from 0 mM or about From 0 mM to 8 mM or about 8 mM, from 0 mM or about 0 mM to 9 mM or about 9 mM, from 0 mM or about 0 mM to 10 mM or about 10 mM, from 0 mM or about 0 mM to 12 mM or about 12 mM, from 0 mM or about 0 mM to 14 mM or about 14 mM, from 0 mM or about 0 mM to 16 mM or about 16 mM, from 0 mM or about 0 mM to 18 mM or about 18 mM, from 0 mM or about 0 mM to 20 mM or about 20 mM.

In some embodiments, the one or more additional components include ethanolamine. In some embodiments, the concentration of ethanolamine is such that after the supplement is combined with the basal medium (such as those described herein), the concentration of ethanolamine in the basal medium is from 0 mg/L or about 0 mg/L, 2 mg/L or about 2 mg/L, 0 mg/L or about 0 mg/L, 4 mg/L or about 4 mg/L, 0 mg/L or about 0 mg/L, 6 mg/L or about 6 mg/L, 0 mg/L or about 0 mg/L, 8 mg/L or about 8 mg/L, 0 mg/L or about 0 mg/L, 10 mg/L or about 10 mg/L, 0 mg/L or about 0 mg/L, 12 mg/L or about 12 mg/L, 0 mg/L or about 0 mg/L, 14 mg/L or about 14 mg/L, 16 mg/L or about 14 mg/L, 18 mg/L or about 14 mg/L, 19 mg/L or about 14 mg/L, 20 mg/L or about 20 mg/L, 21 mg/L or about 20 mg/L, 22 mg/L or about 20 mg/L, 23 mg/L or about 20 mg/L, 24 mg/L or about 20 mg/L, 25 mg/L or about 20 mg/L, 26 mg/L or about 20 mg/L, 27 mg/L or about 20 mg/L, 28 mg/L or about 20 mg/L, 29 mg/L or about 20 mg/L, 30 mg/L or about 30 mg/L, 31 mg/L or about 31 mg/L, 32 mg/L or about 31 mg/L, 33 mg/L or about 31 mg/L, 34 mg/L or about mg/L or about 14 mg/L, 0 mg/L or about 0 mg/L to 16 mg/L or about 16 mg/L, 0 mg/L or about 0 mg/L to 18 mg/L or about 18 mg/L, 0 mg/L or about 0 mg/L to 20 mg/L or about 20 mg/L, 0 mg/L or about 0 mg/L to 22 mg/L or about 22 mg/L, 0 mg/L or about 0 mg/L to 24 mg/L or about 24 mg/L, 0 mg/L or about 0 mg/L to 26 mg/L or about 26 mg/L, 0 mg/L or about 0 mg/L to 28 mg/L or about 28 mg/L, 0 mg/L or about 0 mg/L to 30 mg/L or about 30 mg/L.

In some embodiments, one or more additional components can be provided by adding one or more supplements, e.g., a first supplement and one or more further or additional supplements, to the basal medium.

In some embodiments, the first supplement is prepared by adding or mixing L-glutamine to an existing supplement containing one or more desired components. In some embodiments, L-glutamine is added to or mixed with a serum replacement supplement, e.g., an immune cell serum replacement, e.g., ThermoFisher, #A2598101 or CTS™ immune cell serum replacement. In some embodiments, L-glutamine is added to or mixed with a supplement containing an immune cell serum replacement as described in Smith et al. Clin Transl Immunology. 2015 Jan; 4(1): e31.

In some embodiments, the serum-free medium formulation comprises 90%-98.75% (v/v) or about 90%-98.75% (v/v) of a basal medium and 1.25%-10% (v/v) or about 1.25%-10% (v/v) of a first supplement. In some embodiments, the serum-free medium formulation comprises 90%-97.5% (v/v) or about 90%-97.5% (v/v) of a basal medium and 1.25%-5% (v/v) or about 1.25%-5% (v/v) of a first supplement. In some embodiments, the serum-free medium formulation comprises 95% (v/v) or about 95% (v/v) basal medium and 2.5% ± 0.2% (v/v) or about 2.5% ± 0.2% (v/v), e.g., 2.5% (v/v) or about 2.5% (v/v), of the first supplement. In some embodiments, 1 liter of basal medium is supplemented with 25 milliliters or about 25 milliliters of the first supplement.

In some embodiments, a further supplement, e.g., a second supplement, is combined with the basal medium to provide one or more additional components. In some embodiments, the second supplement includes one or more additional components as described above, including one or more antioxidants, one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, and one or more glucocorticoids. Exemplary components of the second supplement are described above. In some embodiments, the second supplement includes albumin, N-acetylcysteine (NAC) and ethanolamine. In some embodiments, the second supplement includes albumin, N-acetylcysteine (NAC) and ethanolamine, where the concentrations of albumin, NAC and/or ethanolamine are such that after the second supplement is combined with the basal medium (such as those described herein), the concentrations of albumin, NAC and/or ethanolamine are substantially the same as those described herein. In some embodiments, the albumin is human-derived albumin. In some embodiments, the albumin is human-derived albumin from human plasma or serum. In some embodiments, the second supplement is liquid and does not contain or does not contain a significant amount of the free form of glutamine (i.e., L-glutamine). In some embodiments, the second supplement comprises OpTmizer® supplement (part of Thermofisher, A1048503).

In some embodiments, the second supplement is liquid. In some embodiments, the second supplement is not frozen for storage or is not recommended to be frozen. In some embodiments, the serum-free medium formulation comprises about 1.25% to about 5% (v/v), e.g., 2.5% ± 0.2% or about 2.5% ± 0.2%, e.g., 2.5% or 2.6% or about 2.5% or about 2.6% of the second supplement. In some embodiments, one liter of basal medium is supplemented with about 26 milliliters of the second supplement. In some embodiments, the serum-free medium formulation comprises 90% to 97.5% (v/v) or about 90% to about 97.5% (v/v) of the basal medium and 1.25% to 5% (v/v) or about 1.25% to about 5% (v/v) of the second supplement. In some embodiments, the serum-free medium formulation comprises 95% or about 95% (v/v) basal medium and 2.5%±0.2% (v/v) or about 2.5%±0.2% (v/v), e.g., 2.5% (v/v) or 2.6% (v/v) or about 2.5% (v/v) or about 2.6% (v/v) of the second supplement. In some embodiments, 1 liter of basal medium is supplemented with 25 or 26 milliliters or about 25 or 26 milliliters of the second supplement.

In some embodiments, both a first supplement (e.g., a serum replacement supplement, e.g., CTS™ immune cell serum replacement) and an additional supplement (e.g., OpTmizer® cell supplement) are added to the basal medium. In some embodiments, the serum-free medium formulation comprises about 90%-97.5% (v/v) of the basal medium, about 1.25%-5% (v/v) of the first supplement, and about 1.25%-5% (v/v) of the second supplement. In some embodiments, the serum-free medium formulation comprises about 95% (v/v) of the basal medium, about 2.5%±0.2% (v/v) of the first supplement, and about 2.5%±0.2% (v/v) of the second supplement.

In some embodiments, the one or more supplements are concentrated from 2 or about 2 times to 100 times or about 100 times. In some embodiments, the supplements are in a 40 times or about 40 times formulation. In some embodiments, 1 liter of basal medium is supplemented with 20 to 30 milliliters or about 20 to 30 milliliters, e.g., 25±2 milliliters, of at least one supplement comprising a first supplement and optionally one or more additional supplements.

C. Serum-Free Media In some embodiments, the serum-free media comprises a basal medium and a synthetic amino acid (e.g., a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine), a free form of glutamine (i.e., L-glutamine). In some embodiments, the serum-free media comprises a basal medium and a synthetic amino acid (e.g., a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine), a free form of glutamine (i.e., L-glutamine). In some embodiments, the serum-free media further comprises at least one protein or additional component to aid in the maintenance of the T cells, e.g., during the proven process for generating engineered T cells.

In some embodiments, the serum-free medium is a form that contains a synthetic amino acid (e.g., a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine) that can be converted to a free form of glutamine (i.e., L-glutamine) in a cell culture containing cells, where the medium is serum-free. In some embodiments, the synthetic amino acid is soluble in an aqueous solution (e.g., serum-free medium). In some embodiments, the solubility of the synthetic amino acid in an aqueous solution is higher than the free form of glutamine (i.e., L-glutamine). In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the serum-free medium is 0.5 mM to 5 mM or about 0.5 mM to about 5 mM. In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the serum-free medium is 2 mM or about 2 mM. In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) is between 0.5 mM and 1 mM, between 0.5 mM and 1.5 mM, between 0.5 mM and 2 mM, between 0.5 mM and 2.5 mM, between 0.5 mM and 3 mM, between 0.5 mM and 3.5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 4.5 mM, or between 0.5 mM and 5 mM, or between about 0.5 mM and 1 mM, between about 0.5 mM and 1.5 mM, between about 0.5 mM and 2 mM, between about 0.5 mM and 2.5 mM, between about 0.5 mM and 3 mM, between about 0.5 mM and 3.5 mM, between about 0.5 mM and 4 mM, between about 0.5 mM and 4.5 mM, or between about 0.5 mM and 5 mM, inclusive. In some embodiments, the concentration of the dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine) in the serum-free medium is at least 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or at least about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. In some embodiments, the concentration of the dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine, in the serum-free medium is 2 mM or about 2 mM, and the concentration of the free form of L-glutamine in the serum-free medium is 2 mM or about 2 mM.

In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the serum-free medium is about 0.5 mM to about 5 mM. In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the serum-free medium is at or about 2 mM. In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the serum-free medium is between 0.5 mM and 1 mM, between 0.5 mM and 1.5 mM, between 0.5 mM and 2 mM, between 0.5 mM and 2.5 mM, between 0.5 mM and 3 mM, between 0.5 mM and 3.5 mM, between 0.5 mM and 4 mM, between 0.5 mM and 4.5 mM, or between 0.5 mM and 5 mM, or between about 0.5 mM and 1 mM, between about 0.5 mM and 1.5 mM, between about 0.5 mM and 2 mM, between about 0.5 mM and 2.5 mM, between about 0.5 mM and 3 mM, between about 0.5 mM and 3.5 mM, between about 0.5 mM and 4 mM, between about 0.5 mM and 4.5 mM, or between about 0.5 mM and 5 mM, inclusive. In some embodiments, the concentration of the free form of glutamine (i.e., L-glutamine) in the medium is at least 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or at least about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM.

In some embodiments, the serum-free medium comprises at least one protein. In some embodiments, the at least one protein is not of non-mammalian origin. In some embodiments, the at least one protein is human or derived from a human. In some embodiments, the at least one protein is recombinant. In some embodiments, the one or more additional components comprise at least one protein. In some embodiments, the at least one protein is not of non-mammalian origin. In some embodiments, the at least one protein is human or derived from a human. In some embodiments, the at least one protein is recombinant. In some embodiments, the at least one protein comprises albumin, transferrin, insulin, fibronectin, aprotinin or fetuin. In some embodiments, the protein comprises one or more of albumin, insulin or transferrin, optionally one or more of human or recombinant albumin, insulin or transferrin.

In some embodiments, the serum-free medium comprises albumin. In some embodiments, the albumin is derived from a human. In some embodiments, the albumin is derived from human serum or human plasma. In some embodiments, the albumin is recombinant albumin. In some embodiments, the recombinant albumin is derived from a human. In some embodiments, the recombinant albumin is not derived from a human. In some embodiments, the supplement comprises natural albumin. In some embodiments, the natural albumin is derived from a human. In some embodiments, the natural albumin is not derived from a human. In some embodiments, the concentration of albumin in the serum-free medium is from 0 mg/mL or about 0 mg/mL, 2 mg/mL or about 2 mg/mL, 0 mg/mL or about 0 mg/mL, 4 mg/mL or about 4 mg/mL, 0 mg/mL or about 0 mg/mL, 6 mg/mL or about 6 mg/mL, 0 mg/mL or about 0 mg/mL, 8 mg/mL or about 8 mg/mL, 0 mg/mL or about 0 mg/mL, 10 mg/mL or about 10 mg/mL, 0 mg/mL or about 0 mg/mL, 12 mg/mL or about 12 mg/mL, 2 mg/mL or about 2 mg/mL, 4 mg/mL or about 4 mg/mL, 2 mg/mL or about 2 mg/mL, 6 mg/mL or about 6 mg/mL, 2 mg/mL or about 2 mg/mL, 8 mg/mL or about 8 mg/mL, 2 mg/mL or about 2 mg/mL, from 2 mg/mL or about 2 mg/mL, from 12 mg/mL or about 12 mg/mL, from 4 mg/mL or about 4 mg/mL, from 6 mg/mL or about 6 mg/mL, from 4 mg/mL or about 4 mg/mL, from 8 mg/mL or about 8 mg/mL, from 4 mg/mL or about 4 mg/mL, from 10 mg/mL or about 10 mg/mL, from 4 mg/mL or about 4 mg/mL, from 12 mg/mL or about 12 mg/mL, from 6 mg/mL or about 6 mg/mL, from 8 mg/mL or about 8 mg/mL, from 6 mg/mL or about 6 mg/mL, from 10 mg/mL or about 10 mg/mL, from 6 mg/mL or about 6 mg/mL, from 12 mg/mL or about 12 mg/mL, from 8 mg/mL or about 8 mg/mL, from 10 mg/mL or about 10 mg/mL, from 8 mg/mL or about 8 mg/mL, from 12 mg/mL or about 12 mg/mL mg/mL, 10 mg/mL or about 10 mg/mL to 12 mg/mL or about 12 mg/mL, or 10 mg/mL or about 10 mg/mL to 15 mg/mL or about 15 mg/mL, inclusive. In some embodiments, the albumin in the medium is 5 mg/mL or about 5 mg/mL.

In some embodiments, the serum-free medium includes transferrin or a transferrin substitute (such as those described herein). In some embodiments, the transferrin or transferrin substitute is derived from a human. In some embodiments, the transferrin or transferrin substitute is derived from human serum or plasma. In some embodiments, the concentration of transferrin in the serum-free medium is from 10 mg/L or about 10 mg/L to 50 mg/L or about 50 mg/L, 10 mg/L or about 10 mg/L to 100 mg/L or about 100 mg/L, 10 mg/L or about 10 mg/L to 150 mg/L or about 150 mg/L, 10 mg/L or about 10 mg/L to 200 mg/L or about 200 mg/L, 10 mg/L or about 10 mg/L to 250 mg/L or about 250 mg/L, 10 mg/L or about 10 mg/L to 300 mg/L or about 300 mg/L, 10 mg/L or about 10 mg/L to 350 mg/L. or from about 350 mg/L, 10 mg/L or about 10 mg/L, 400 mg/L or about 400 mg/L, 10 mg/L or about 10 mg/L, 450 mg/L or about 450 mg/L, 10 mg/L or about 10 mg/L, 500 mg/L or about 500 mg/L, 10 mg/L or about 10 mg/L, 550 mg/L or about 550 mg/L, 10 mg/L or about 10 mg/L, 600 mg/L or about 600 mg/L, 10 mg/L or about 10 mg/L, 650 mg/L or about 650 mg/L, or 10 mg/L or about 10 mg/L, 750 mg/L or about 750 mg/L. In some embodiments, the concentration of transferrin in the serum-free medium is 100 mg/L or about 100 mg/L. In some embodiments, the concentration of transferrin in the serum-free medium is at or about 50 mg/L to 150 mg/L.

In some embodiments, the supplement comprises insulin or an insulin substitute (such as those described herein). In some embodiments, the insulin is derived from a human. In some embodiments, the insulin is recombinant insulin. In some embodiments, the insulin is recombinant human insulin. In some embodiments, the concentration of insulin (or insulin substitute) in the serum-free medium is from 1 mg/L or about 1 mg/L, 2.5 mg/L or about 2.5 mg/L, 1 mg/L or about 1 mg/L, 5 mg/L or about 5 mg/L, 1 mg/L or about 1 mg/L, 7.5 mg/L or about 7.5 mg/L, 1 mg/L or about 1 mg/L, 10 mg/L or about 10 mg/L, 1 mg/L or about 1 mg/L, 12.5 mg/L or about 12.5 mg/L, 1 mg/L or about 1 mg/L, 15 mg/L or about 15 mg/L, 1 mg/L or about 1 mg/L, 17.5 mg/L or about 17.5 mg/L, 1 mg/L or about 1 mg/L, 20 mg/L or about 20 mg/L, 1 mg/L or about 1 mg/L, 1 mg/L or about 22.5 mg/L, 1 mg/L or about 1 mg/L to 25 mg/L or about 25 mg/L, 1 mg/L or about 1 mg/L to 27.5 mg/L or about 27.5 mg/L, or 1 mg/L or about 1 mg/L to 30 mg/L or about 30 mg/L. In some embodiments, the concentration of insulin or insulin substitute in the serum-free medium is 10 mg/L or about 10 mg/L. In some embodiments, the concentration of insulin or insulin substitute in the serum-free medium is 7.5 mg/L or about 7.5 mg/L to 12.5 mg/L or about 12.5 mg/L.

In some embodiments, the serum-free medium does not contain phenol red. In some embodiments, the serum-free medium contains phenol red.

In some embodiments, the serum-free medium comprises a nutrient mixture of inorganic salts, sugars, amino acids, and may optionally contain vitamins, organic acids, antioxidants, lipids, growth factors, N-acetylcysteine, ethanolamine, and/or buffers. Examples include those described herein, such as in the sections above, including inorganic salts, sugars, amino acids, vitamins, organic acids, antioxidants, lipids, growth factors, N-acetylcysteine, ethanolamine, and/or buffers.

In some embodiments, the serum-free medium comprises one or more components selected from one or more of: one or more antioxidants, one or more albumins or albumin substitutes, one or more lipid agents, one or more insulins or insulin substitutes, one or more transferrins or transferrin substitutes, one or more trace elements, one or more glucocorticoids, one or more inorganic salts, one or more energy sources, one or more buffering agents, one or more pyruvates, one or more pH indicators, one or more amino acids, and one or more vitamins. In some embodiments, the antioxidant is selected from the group consisting of N-acetyl-L-cysteine, 2-mercaptoethanol, and D,L-tocopherol acetate, or derivatives or mixtures thereof. In some embodiments, the albumin is human serum albumin. In some embodiments, the lipid agents are Human Ex-Cyte® and ethanolamine. In some embodiments, the insulin is human zinc insulin. In some embodiments, the transferrin is human iron-saturated transferrin. In some embodiments, the glucocorticoid is hydrocortisone. In some embodiments, the inorganic salt component comprises one or more inorganic salts selected from the group consisting of one or more calcium salts, one or more potassium salts, one or more magnesium salts, one or more sodium salts, one or more carbonate salts, and one or more phosphate salts. In some embodiments, the energy source is D-glucose. In some embodiments, the buffer is HEPES. In some embodiments, the pyruvate salt is sodium pyruvate. In some embodiments, the pH indicator is phenol red. In some embodiments, the amino acid component comprises one or more amino acids selected from the group consisting of glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine HCL, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and salts and derivatives thereof. In some embodiments, the vitamin component comprises one or more vitamins selected from the group consisting of biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, pyridoxal HCl, riboflavin, thiamine HCl, and vitamin B12 and its derivatives. In some embodiments, the components are N-acetyl-L-cysteine, 2-mercaptoethanol, human serum albumin, D,L-tocopherol acetate, Human Ex-Cyte®, ethanolamine, human zinc insulin, iron-saturated transferrin, Se4 ⁺ , hydrocortisone, Ca2 ⁺ , K ⁺ , Mg2 ⁺ , Na ⁺ , _CO32- , ^PO43- , D-glucose, HEPES, sodium pyruvate _, phenol red, glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic ^acid , L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine HCL, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine ... -Tryptophan, L-Tyrosine, and L-Valine, Biotin, D-Calcium Pantothenate, Choline Chloride, Folic Acid, i-Inositol, Niacinamide, Pyridoxal HCl, Riboflavin, Thiamine HCl, and Vitamin B12.

In some embodiments, a serum-free medium is provided that includes a basal medium and at least one supplement. Various examples of basal media and supplements are described herein, such as in the sections above.

In some embodiments, the serum-free medium formulation comprises 90% or about 90% to 97.5% or about 97.5% (v/v) of the basal medium, 2.5% or about 2.5% to 10% or about 10% (v/v) of a supplement, such as a first supplement and/or a second supplement. In some embodiments, the serum-free medium formulation comprises 90% or about 90% to 97.5% or about 97.5% (v/v) of the basal medium, 1.25% or about 1.25% to 5% or about 5% (v/v) of the first supplement, and 1.25% or about 1.25% to 5% or about 5% (v/v) of the second supplement.

In some embodiments, the serum-free medium comprises a basal medium supplemented with one or more supplements, such as OpTmizer™ T Cell Expansion Basal Medium (ThermoFisher). In some embodiments, the one or more supplements are serum-free. In some embodiments, the serum-free medium comprises a basal medium supplemented with a supplement for the maintenance of cells (e.g., T cells), such as OpTmizer™ T Cell Expansion Supplement (ThermoFisher). In some embodiments, the serum-free medium further comprises a free form of an amino acid, such as L-glutamine. In some embodiments, the serum-free medium does not contain a recombinant cytokine, such as one or more of recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15. In certain embodiments, the serum-free medium can be used for any one or more steps of the processes described herein, such as one or more steps described in section I. In some embodiments, such serum-free medium is used during incubation and/or harvesting, recovery or compounding of the cells.

In some embodiments, the serum-free medium comprises a basal medium supplemented with a T cell supplement and a free form of L-glutamine. In some embodiments, the serum-free medium comprises OpTmizer™ T cell expansion supplement and OpTmizer™ T cell expansion basal medium supplemented with L-glutamine. In some embodiments, the serum-free medium comprises about 2.6% OpTmizer™ T cell expansion supplement and OpTmizer™ T cell expansion basal medium supplemented with about 1.0% L-glutamine (final concentration of about 2 mM). In some embodiments, such serum-free medium does not contain recombinant cytokines such as one or more of recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15. In certain embodiments, the serum-free medium can be used for any one or more steps of the processes described herein, such as one or more steps described in section I. In some embodiments, such serum-free medium is used during incubation and/or harvesting, recovery or compounding of the cells.

In some embodiments, the serum-free medium comprises a basal medium supplemented with a supplement for the maintenance of cells (e.g., T cells), such as OpTmizer™ T cell expansion and proliferation supplement (ThermoFisher), and further comprises a serum replacement supplement, such as an immune cell serum replacement, such as ThermoFisher, #A2596101, CTS™ immune cell serum replacement, or an immune cell serum replacement as described in Smith et al. Clin Transl Immunology. 2015 Jan; 4(1): e31. In some embodiments, the serum-free medium further comprises a free form of an amino acid, such as L-glutamine. In some embodiments, the serum-free medium further comprises a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), such as the dipeptide in Glutamax™ (ThermoFisher). In some embodiments, the serum-free medium comprises one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In certain embodiments, the one or more cytokines are expressed by T cells and/or bind and/or can bind to a receptor endogenous to T cells. In particular embodiments, the one or more cytokines are or include members of the four-alpha helix bundle family of cytokines. In some embodiments, members of the four-alpha helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the serum-free medium further comprises one or more of recombinant human IL-2, recombinant human IL-7, and/or recombinant human IL-15. In particular embodiments, the serum-free medium can be used for any one or more steps of the processes described herein, such as one or more steps described in section I. In certain embodiments, serum-free media can be used for any one or more steps including sample preparation, selection, stimulation and/or manipulation.

In some embodiments, the serum-free medium comprises basal medium supplemented with T cell supplement, immune cell serum replacement, free form of L-glutamine, dipeptide form of L-glutamine, recombinant IL-2, recombinant IL-7, and/or recombinant IL-15. In some embodiments, the serum-free medium comprises OpTmizer™ T cell expansion supplement, CTS™ immune cell serum replacement, L-glutamine, L-alanyl-L-glutamine, recombinant human IL-2, recombinant human IL-7, and recombinant human IL-15. In some embodiments, the serum-free medium comprises OpTmizer™ T Cell Expansion Basal Medium supplemented with about 2.6% OpTmizer™ T Cell Expansion Supplement, about 2.5% CTS™ Immune Cell Serum Replacement, about 1.0% L-glutamine (final concentration of about 2 mM), about 1.0% L-alanyl-L-glutamine (final concentration of about 2 mM), about 100 IU/mL recombinant human IL-2, about 600 IU/mL recombinant human IL-7, and about 100 IU/mL recombinant human IL-15. In certain embodiments, the serum-free medium can be used for any one or more steps of the processes described herein, such as one or more steps described in section I. In certain embodiments, the serum-free medium can be used for any one or more steps including sample preparation, selection, stimulation, and/or manipulation.

In some embodiments, the serum-free medium is a concentrated medium formulation. In some embodiments, the serum-free medium is not a concentrated medium formulation. In some embodiments, the serum-free medium is concentrated from at or about 2-fold to at or about 100-fold. In some embodiments, the serum-free medium is a 10-fold or about 10-fold formulation. In some embodiments, the serum-free medium can be stored at or about 2°C to 8°C.

IV. Recombinant Proteins In some embodiments, the cells treated, processed, manipulated and/or produced by the methods provided herein contain or express, or are engineered to contain or express, a recombinant protein, such as a recombinant receptor, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In certain embodiments, the methods provided herein have the ability to produce and/or produce cells, or a population or composition that contains and/or is enriched for cells. These cells are engineered to express or contain a recombinant protein. In various embodiments, engineered, transformed, transduced, transfected cells, such as immune cells, for example T cells, that express one or more recombinant proteins are provided. In certain embodiments, at least one of the one or more recombinant proteins is a recombinant receptor, such as a receptor that contains an antigen receptor and one or more of its components.

A. Recombinant Receptors In some embodiments, engineered cells, such as immune cells, e.g., T cells, are provided that express one or more recombinant receptors. The receptor is a receptor that contains an antigen receptor and one or more of its components. The recombinant receptor can include chimeric receptors, such as receptors that contain a ligand-binding domain or a binding fragment thereof and an intracellular signaling domain or region, functional non-TCR antigen receptors, chimeric antigen receptors (CARs), T cell receptors (TCRs), e.g., recombinant TCRs or genetically modified TCRs, chimeric autoantibody receptors (CAARs), and components of any of the above. Recombinant receptors, e.g., CARs, generally contain an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, which in some aspects are linked via a linker and/or transmembrane domain(s). In some embodiments, the engineered cells express two or more receptors that contain different components, domains, or regions. In some aspects, the two or more receptors allow for spatial or temporal control, or control of the specificity, activity, antigen (or ligand) binding, function, and/or expression of the recombinant receptor.

1. Chimeric Antigen Receptor (CAR)
In some embodiments of the provided methods, the chimeric receptor, e.g., chimeric antigen receptor, contains one or more domains that combine a ligand binding domain (e.g., an antibody or antibody fragment) that confers specificity for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion that provides a primary activation signal, e.g., a T cell activation domain. In some embodiments, the intracellular signaling domain contains, or additionally contains, a costimulatory signaling domain to enhance effector function. In some embodiments, the chimeric receptor, when engineered into immune cells, can regulate the activity of T cells and, in some cases, regulate the differentiation or homeostasis of T cells, thereby resulting in engineered cells with improved in vivo longevity, survival, and/or persistence, e.g., for use in adoptive cell therapy.

Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described in, e.g., International Patent Application Publication Nos. 200014257, 2013126726, 2012/129514, 2014031687, 2013/166321, 2013/071154, and 2013/123061; U.S. Patent Application Publication Nos. 2002131960, 2013287748, 20130149337, and U.S. Pat. Those described in Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and European Patent Application No. 2 537 416, and/or Sadelain et al., Cancer Discov. 2013 April;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October;24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, antigen receptors include CARs described in U.S. Pat. No. 7,446,190 and those described in International Patent Publication No. WO2014055668 A1. Examples of CARs include those disclosed in any of the above-mentioned publications, e.g., WO 2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, 2013/0149337, U.S. Pat. No. 7,446,190, and 8,389,282; Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9):689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO 2014031687, U.S. Patent Nos. 8,339,645, 7,446,179, 2013/0149337, 7,446,190, and 8,389,282.

Chimeric receptors such as CARs are typically portions of antibody molecules, and contain the extracellular antigen-binding domain, such as the generic variable heavy (VH) and/or variable light (VL) chain regions of an antibody, e.g., an scFv antibody fragment.

In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a sugar molecule or another molecule. In some embodiments, the antigen is selectively expressed or overexpressed on diseased or condition cells, e.g., tumor or pathogenic cells, compared to standard target cells or tissues or non-standard target cells or tissues. In another embodiment, the antigen is expressed on normal cells and/or engineered cells.

In some embodiments, the antigen is selected from the group consisting of αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5, also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinase er b-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3 , MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), nervous system cell adhesion molecule (NCAM), oncofetal antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein The antigen targeted by the receptor may be or include: protein 72 (TAG72), tyrosinase-related protein 1 (also known as TRP1, TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and/or a biotinylated molecule, and/or a molecule expressed by HIV, HCV, HBV or other pathogens. The antigen targeted by the receptor may in some embodiments include an antigen associated with a B-cell malignancy, such as any of a number of known B-cell markers. In some embodiments, the antigen is or comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30. In some embodiments, the antigen is or comprises a pathogen-specific antigen or a pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and/or a parasitic antigen.

に示される配列を有するか、またはこれからなるリンカーを含む。 In some embodiments, the antibody is an antigen-binding fragment that includes one or more linkers linking two antibody domains or domains, such as, for example, a heavy chain variable ( _VH ) region and a light chain variable ( _VL ) region, such as, for example, an scFv. The linker is usually, for example, a flexible peptide linker and/or a soluble peptide linker. The linker is rich in glycine and serine, and/or optionally, threonine. In some embodiments, the linker further comprises a charged residue, such as lysine and/or glutamate, which may improve solubility. In some embodiments, the linker further comprises one or more prolines. In some aspects, the linker rich in glycine and serine (and/or threonine) comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of such amino acid(s). In some embodiments, they comprise at least or at least about 50%, 55%, 60%, 70% or 75% glycine, serine, and/or threonine. In some embodiments, the linker is substantially entirely composed of glycine, serine, and/or threonine. Linkers are generally between about 5 and about 50 amino acids in length, usually 10 or about 10 to 30 or about 30 amino acids in length, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and in some instances 10-25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO:122) or GGGS (3GS; SEQ ID NO:123), for example, 2, 3, 4 to 5 repeats of such sequences. Exemplary linkers include

The linker comprises a linker having or consisting of the sequence shown in

The antigen targeted by the receptor, in some embodiments, includes an antigen associated with a B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

In some embodiments, the antigen or antigen binding domain is CD19. In some embodiments, the scFv contains a VH and a VL from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds CD19 is a murine-derived antibody, e.g., FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. 2016/0152723.

The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, such as intact antibodies and functional (antigen-binding) antibody fragments, such as Fab (Fragment antigen binding) fragments, F(ab') ₂ fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable ( _VH ) regions capable of specifically binding to an antigen, single chain antibody fragments, such as single chain variable fragments (scFv) and single domain antibody (e.g. sdAb, sdFv, nanobody) fragments. The term also includes engineered and/or otherwise modified immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g. bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof, also referred to herein as "antigen-binding fragments." The term also encompasses intact or full-length antibodies, of any class or subclass, such as IgG and its subclasses, IgM, IgE, IgA, and IgD.

"Complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous amino acid sequences within an antibody variable region that confer antigen specificity and/or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).

The exact amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (Chothia numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topograph”, J. Mol. Biol. 262, 732-745 (Contact numbering scheme), Lefranc MP et al., “IMGT unique numbering for immunoglobulin and Tcell receptor variable domains and Ig superfamily V-like domains”, Dev Comp Immunol, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme), and Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70 (the "Aho" numbering scheme), and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm", PNAS, 1989,86(23):9268-9272 (the "AbM" numbering scheme).

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions, adjusted by insertion strings such as "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("insertions") at different positions, which results in different numbering. The Contact scheme is based on the analysis of complex crystal structures, which are similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used in the AbM antibody modeling software by Oxford Molecular.

Table 1 below lists exemplary position boundaries for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by the Kabat, Chothia, AbM and Contact schemes, respectively. For CDR-H1, the residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs, e.g., FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, and so on. Note that since the Kabat numbering scheme shown places the insertion at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention shown varies between H32 and H34 depending on the loop length.

Table 1. CDR boundaries according to various numbering schemes

Thus, unless otherwise specified, the "CDR" or "complementarity determining region" or the designated individual CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof, e.g., the variable region thereof, should be understood to encompass the complementarity determining region (or specific complementarity determining region) defined by any of the above schemes or another known scheme. For example, if a particular CDR (e.g., CDR-H3) in a given _VH or _VL region amino acid sequence is stated to contain the amino acid sequence of the corresponding CDR, such CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region defined by any of the above schemes or another known scheme. In some embodiments, a specific CDR sequence is specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes, although it is understood that the provided antibodies may include CDRs described according to another numbering scheme described above or another numbering scheme known to those of skill in the art.

Similarly, unless otherwise specified, a given antibody or region thereof, such as the FRs of its variable regions or the individual FRs specified (e.g., FR-H1, FR-H2, FR-H3, FR-H4), should be understood to encompass the framework regions (or specified framework regions) defined by any of the known schemes. In some instances, a scheme for identifying a particular CDR, FR or FRs or CDRs is specified, such as CDRs defined by the Kabat, Chothia, AbM or Contact methods, or other known schemes. In other cases, the specific amino acid sequences of the CDRs or FRs are given.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of native antibodies ( _VH and _VL , respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single _VH or _VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or _VH domains, respectively, with a _VH or _VL domain from an _antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The antibody contained in the provided CAR is an antibody fragment. An "antibody fragment" or an "antibody binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen that the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, single-chain antibody molecules such as heavy chain variable ( _VH ) regions, e.g., scFvs, single-domain antibodies containing only the _VH region, and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding domain in the provided CAR is or contains an antibody fragment containing a variable heavy chain ( _VH ) region and a variable light chain ( _VL ) region. In certain embodiments, the antibody is a single-chain antibody fragment containing a heavy chain variable ( _VH ) region and/or a light chain variable ( _VL ) region, e.g., scFv.

In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells expressing CD19 of human origin (Ling, NR, et al. (1987) Leucocyte typing III.302). In some embodiments, the FMC63 antibody comprises CDRH1 and CDRH2 as shown in SEQ ID NO:51 and SEQ ID NO:52, respectively, CDRH3 as shown in SEQ ID NO:53 or SEQ ID NO:54, and CDRL1 as shown in SEQ ID NO:55 and CDR L2 as shown in SEQ ID NO:55 or SEQ ID NO:57, and CDR L3 as shown in SEQ ID NO:58 or SEQ ID NO:59. In some embodiments, the FMC63 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (V _L ) comprising the amino acid sequence of SEQ ID NO:61.

In some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:55, the CDRL2 sequence of SEQ ID NO:56, and the CDRL3 sequence of SEQ ID NO:58, and/or a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:51, the CDRH2 sequence of SEQ ID NO:52, and the CDRH3 sequence of SEQ ID NO:53. In some embodiments, the scFv comprises a variable heavy chain region depicted in SEQ ID NO:60 and a variable light chain region depicted in SEQ ID NO:61. In some embodiments, the variable heavy chain and the variable light chain are joined by a linker. In some embodiments, the linker is depicted in SEQ ID NO:62. In some embodiments, the scFv comprises, in order, a _VH , a linker, and a _VL . In some embodiments, the scFv comprises, in order, a _VL , a linker, and a _VH . In some embodiments, the scFv is encoded by a sequence of nucleotides set forth in SEQ ID NO:63, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:63. In some embodiments, the scFv comprises a sequence of amino acids set forth in SEQ ID NO:64, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:64.

In some embodiments, the scFv is derived from SJ25C1. SJ25C1 refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells expressing CD19 of human origin (Ling, NR, et al. (1987) Leucocyte typing III.302). In some embodiments, the SJ25C1 antibody comprises CDRH1, CDRH2, CDRH3 as set forth in SEQ ID NOs:65-67, respectively, and comprises CDRL1, CDRL2 and CDRL3 sequences as set forth in SEQ ID NOs:68-70, respectively. In some embodiments, the SJ25C1 antibody comprises a heavy chain variable region (V _H ) comprising the amino acid sequence of SEQ ID NO:71 and a light chain variable region (V _L ) comprising the amino acid sequence of SEQ ID NO:72.

In some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:73, the CDRL2 sequence of SEQ ID NO:74, and the CDRL3 sequence of SEQ ID NO:75, and/or a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:76, the CDRH2 sequence of SEQ ID NO:77, and the CDRH3 sequence of SEQ ID NO:78. In some embodiments, the scFv comprises a variable heavy chain region depicted in SEQ ID NO:71 and a variable light chain region depicted in SEQ ID NO:72. In some embodiments, the variable heavy chain and the variable light chain are joined by a linker. In some embodiments, the linker is depicted in SEQ ID NO:79. In some embodiments, the scFv comprises, in order, a _VH , a linker, and a _VL . In some embodiments, the scFv comprises, in order, a _VL , a linker, and a _VH . In some embodiments, the scFv comprises a sequence of amino acids as shown in SEQ ID NO:80, or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:80.

In some embodiments, the antibody or antigen-binding fragment (e.g., scFv or _VH domain) specifically recognizes an antigen such as BCMA. In some embodiments, the antibody or antigen-binding fragment is derived from or is a variant of an antibody or antigen-binding fragment that specifically binds BCMA.

In some embodiments, the CAR is specific for BCMA, e.g., an anti-BCMA CAR, such as human BCMA. Chimeric antigen receptors containing anti-BCMA antibodies, including mouse anti-human BCMA antibodies and human anti-human antibodies, and cells expressing such chimeric receptors have been previously described. See Carpenter et al., Clin Cancer Res., 2013, 19(8):2048-2060, WO 2016/090320, WO 2016090327, WO 2010104949A2, and WO 2017173256. In some embodiments, the antigen or antigen binding domain is BCMA. In some embodiments, the scFv contains a VH and a VL from an antibody or antibody fragment specific for BCMA. In some embodiments, the antibody or antibody fragment that binds BCMA is or contains a VH and VL from an antibody or antibody fragment set forth in International Patent Application Publication Nos. 2016/090327 and 2016/090320.

In some embodiments, the anti-BCMA CAR contains an antigen binding domain, such as an scFv, that contains a variable heavy ( _VH ) region and/or a variable light ( _VL ) region derived from an antibody described in WO2016/090320 or WO2016090327. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:116 and a _VL as set forth in SEQ ID NO:117. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:118 and a _VL as set forth in SEQ ID NO:119. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:120 and a _VL as set forth in SEQ ID NO:121. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:113 and a _VL as set forth in SEQ ID NO:114. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:125 and a _VL as set forth in SEQ ID NO:126. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:127 and a _VL as set forth in SEQ ID NO:128. In some embodiments, the antigen binding domain, such as an scFv, contains a _VH as set forth in SEQ ID NO:129 and a _VL as set forth in SEQ ID NO:130. In some embodiments, _{the VH or VL} has a sequence of _{amino acids} that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing VH or VL sequences and retains binding to BCMA. In some embodiments, the _VH region is amino terminal to the _VL region. In some embodiments, the _VH region is carboxy terminal to the _VL region.

In some embodiments, the antigen or antigen binding domain is GPRC5D. In some embodiments, the scFv contains a VH and a VL from an antibody or an antibody fragment specific for GPRC5D. In some embodiments, the antibody or antibody fragment that binds to GPRC5D is or contains a VH and a VL from an antibody or antibody fragment set forth in International Patent Application Publication Nos. 2016/090329 and 2016/090312.

In some aspects, the CAR comprises a ligand (e.g., antigen) binding domain that binds to or recognizes, e.g., specifically recognizes, a universal tag or universal epitope. In some aspects, the binding domain can bind to a molecule, tag, polypeptide, and/or epitope that can be linked to a different binding molecule (e.g., antibody or antigen-binding fragment) that recognizes an antigen associated with a disease or disorder. Exemplary tags or epitopes include dyes (e.g., fluorescein isothiocyanate) or biotin. In some aspects, a binding molecule (e.g., antibody or antigen-binding fragment) linked to a tag that recognizes an antigen associated with a disease or disorder, e.g., a tumor antigen, together with an engineered cell expressing a CAR specific for the tag, provides cytotoxicity or other effector function of the engineered cell. In some aspects, the specificity of the CAR for an antigen associated with a disease or disorder is provided by a tagged binding molecule (e.g., antibody), and different tagged binding molecules can be used to target different antigens. Exemplary CARs specific for universal tags or universal epitopes include those described, for example, in U.S. Pat. No. 9,233,125, WO2016/030414, Urbanska et al., (2012) Cancer Res 72: 1844-1852, and Tamada et al., (2012). Clin Cancer Res 18:6436-6445.

In some embodiments, the antigen is or comprises a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (e.g., viral antigen from HIV, HCV, HBV, etc.), bacterial antigen, and/or parasitic antigen. In some embodiments, the CAR comprises a TCR-like antibody, e.g., an antibody or fragment (e.g., scFv), that specifically recognizes an intracellular antigen, e.g., a tumor-associated antigen, presented on the cell surface as a major histocompatibility complex (MHC)-peptide complex. In some embodiments, an antibody or antigen-binding portion thereof that recognizes an MHC-peptide complex can be expressed on a cell as part of a recombinant receptor, e.g., an antigen receptor. Among the antigen receptors are functional non-T cell receptor (TCR) antigen receptors, e.g., chimeric antigen receptors (CARs). In some embodiments, a CAR comprising an antibody or antigen-binding fragment that exhibits TCR-like specificity for a peptide-MHC complex may also be referred to as a TCR-like CAR. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, that is recognized on the cell surface in association with an MHC molecule as in a TCR. In some embodiments, the extracellular antigen-binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via a linker and/or a transmembrane domain. In some embodiments, such molecules can typically mimic or approximate signals through natural antigen receptors, such as TCRs, and optionally through such receptors in combination with costimulatory receptors.

Reference to "major histocompatibility complex" (MHC) refers to a protein, typically a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can optionally complex with a polypeptide peptide antigen (including peptide antigens processed by intracellular machinery). In some cases, MHC molecules can be displayed or expressed on the cell surface, e.g., as a complex with a peptide, i.e., an MHC-peptide complex, to present the antigen in a conformation that is recognized by an antigen receptor on a T cell, such as a TCR, or a TCR-like antibody. Generally, MHC class I molecules are heterodimers, with a transmembrane α chain, optionally containing three α domains, and a non-covalently associated β2 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which usually span the membrane. MHC molecules can include an effective portion of MHC that includes the antigen binding site(s) for binding peptides and the sequences necessary for recognition by the appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides from the cytosol to the cell surface; in this case, MHC-peptide complexes are recognized by T cells, for example, generally CD8 ⁺ T cells, but occasionally CD4+ T cells. In some embodiments, MHC class II molecules deliver peptides from the vesicular system to the cell surface; in this case, they are usually recognized by CD4 ⁺ T cells. In general, MHC molecules are encoded by a group of linked loci collectively referred to as H-2 in mice and human leukocyte antigens (HLA) in humans. Therefore, in general, human MHC may also be referred to as human leukocyte antigens (HLA).

The term "MHC-peptide complex" or "peptide-MHC complex" or variations thereof generally refers to a complex or association of a peptide antigen with an MHC molecule, such as by non-covalent interaction of a peptide housed in a binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or presented on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, e.g., a TCR, a TCR-like CAR, or an antigen-binding portion thereof.

In some embodiments, a peptide, such as a peptide antigen or epitope of a polypeptide, can associate with an MHC molecule, for example for recognition by an antigen receptor. Generally, the peptide is derived from or based on a fragment of a longer biomolecule, such as a polypeptide or protein. In some embodiments, the peptide is typically about 8 to about 24 amino acids in length. In some embodiments, the peptide has a length of 9 to 22 or about 9 to 22 amino acids for recognition at an MHC class II complex. In some embodiments, the peptide has a length of 8 to 13 or about 8 to 13 amino acids for recognition at an MHC class I complex. In some embodiments, upon recognition of the peptide in the context of an MHC molecule, such as an MHC-peptide complex, an antigen receptor, such as a TCR or TCR-like CAR, provides or triggers an activation signal to the T cell that induces a T cell response, for example, T cell proliferation, cytokine production, a cytotoxic T cell response or other response.

In some embodiments, the TCR-like antibodies or antigen-binding portions are known or can be made by known methods (see, e.g., U.S. Patent Application Publication Nos. US 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850; US 2007/00992530; US20090226474; US20090304679; and Application Publication No. WO 03/068201).

In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be generated by immunizing a host with an effective amount of an immunogen that includes a particular MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen that can bind to MHC, such as a tumor antigen, such as a universal tumor antigen, a myeloma antigen, or other antigens described below. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response; in which case the immunogen retains its three-dimensional form for a period of time sufficient to elicit an immune response to the three-dimensional representation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine whether the desired antibodies have been produced that recognize the three-dimensional representation of the peptide in the binding groove of the MHC molecule. In some embodiments, the antibodies produced can be assayed to confirm that the antibodies can distinguish the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and a complex of the MHC with an unrelated peptide. The desired antibodies can then be isolated.

In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be obtained by using antibody library display methods, such as phage antibody libraries. In some embodiments, phage display libraries of mutant Fab, scFv, or other antibody forms are generated, e.g., whose members are mutated at one or more residues in the CDR(s). See, e.g., U.S. Patent Application Publication Nos. US20020150914; US2014/0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

In some embodiments, the antigen is CD20. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for CD20. In some embodiments, the antibody or antibody fragment that binds to CD20 is rituximab or is derived from rituximab, e.g., an scFv of rituximab.

In some embodiments, the antigen is CD22. In some embodiments, the scFv contains a VH and a VL from an antibody or antibody fragment specific for CD22. In some embodiments, the antibody or antibody fragment that binds to CD22 is m971 or is derived from m971, e.g., an scFv of m971.

In some embodiments, the chimeric antigen receptor comprises an extracellular portion that contains an antibody or an antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion that contains an antibody or a fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv.

In some embodiments, the antibody portion of the recombinant receptor, e.g., CAR, further comprises at least a portion of an immunoglobulin constant region, e.g., a hinge region, e.g., an IgG4 hinge region and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or constant portion is of a human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition component, e.g., an scFv, and the transmembrane domain. The spacer can be of a length that provides increased cellular responsiveness following antigen binding compared to the absence of the spacer. Exemplary spacers are described, but are not limited to, in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, International Patent Application Publication No. 2014031687, U.S. Patent No. 8,822,647, or U.S. Patent Publication No. 2014/0271635.

In some embodiments, the constant region or constant portion is of a human IgG, e.g., IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (set forth in SEQ ID NO:81) and is encoded by the sequence set forth in SEQ ID NO:82. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:83. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:84. In some embodiments, the constant region or constant portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:85. In some embodiments, the spacer has a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85. In some embodiments, the spacer has a sequence as set forth in SEQ ID NOs: 86-94. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 86-94.

In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to an extracellular domain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is typically linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex in the case of a CAR, and/or signaling through another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., an scFv) and the intracellular signaling domain. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane domains and an intracellular signaling domain.

In one embodiment, a transmembrane domain that naturally associates with one of the domains in a receptor, e.g., CAR, is used. In some examples, the transmembrane domain is selected or modified by amino acid substitutions to avoid binding of such a domain to a transmembrane domain of the same or a different surface membrane protein, minimizing interactions with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain is derived in some aspects from any membrane-bound or transmembrane protein. The transmembrane region includes those derived from the alpha, beta, or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., including at least the transmembrane region thereof). Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain contains primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains the transmembrane portion of CD28.

In some embodiments, the extracellular domain and the transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any described herein. In some embodiments, the receptor contains the extracellular portion of the molecule from which the transmembrane domain is derived, such as the extracellular portion of CD28.

The intracellular signaling domain mimics or approximates a signal through a natural antigen receptor, a signal through a receptor integrated with a costimulatory receptor, and/or a signal through only a costimulatory receptor. In some embodiments, a short oligo- or polypeptide linker, e.g., one that contains glycine and serine, e.g., a glycine-serine doublet, e.g., a linker that is between 2 and 10 amino acids in length, is present between the transmembrane domain and the cytoplasmic signaling domain of the CAR to form a bond therebetween.

T cell activation, in some aspects, is described as being mediated by two types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, CARs contain one or both of these signaling components.

Receptors, such as CARs, generally contain at least one intracellular signaling component or multiple intracellular signaling components. In some aspects, CARs contain a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. The primary cytoplasmic signaling sequence that acts stimulatory can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAMs that contain primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as the TCR CD3 chain, which mediates T cell activation and cytotoxicity, e.g., the CD3 zeta chain. Thus, in some aspects, the antigen binding portion is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or another CD3 transmembrane domain. In some embodiments, the receptor, e.g., a CAR, further comprises a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, a CAR or another chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor gamma and CD8, CD4, CD25, or CD16.

In some embodiments, upon ligation of the CAR or another chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of a standard effector function or response of an immune cell, such as, for example, a T cell engineered to express the CAR. For example, in some situations, the CAR includes a function of a T cell, such as cytolytic activity or T-helper activity, such as secretion of a cytokine or another factor. In some embodiments, a truncated portion of an antigen receptor component or intracellular signaling domain of a costimulatory molecule is used in place of an intact immunostimulatory chain, for example, where it transmits an effector function signal. In some embodiments, the one or more intracellular signaling domains include a cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also include a cytoplasmic sequence of a co-receptor that cooperates with such receptor in the natural context to initiate signal transduction following ligation of the antigen receptor.

In the context of a native TCR, full activation generally requires not only signaling through the TCR but also a costimulatory signal. Thus, in some embodiments, components for generating a secondary or costimulatory signal are also included in the CAR to drive full activation. In other embodiments, the CAR does not include components for generating a costimulatory signal. In some aspects, additional CARs are expressed in the same cell to provide components for generating a secondary or costimulatory signal.

In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR contains a signaling domain and/or a transmembrane portion of a costimulatory receptor, e.g., CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR contains both an activating component and a costimulatory component. In some embodiments, the chimeric antigen receptor contains an intracellular domain or a functional variant thereof from a T cell costimulatory molecule, e.g., between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

In some embodiments, the activation domain is included within one CAR, while the costimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR includes an activating CAR or a stimulatory CAR, a costimulatory CAR, both expressed on the same cell (see WO 2014/055668). In some aspects, the cell includes one or more stimulatory CARs or an activating CAR and/or a costimulatory CAR. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), e.g., a CAR that recognizes an antigen other than an antigen associated with and/or specific for a disease or condition, such that the activation signal delivered via the disease-targeted CAR is reduced or inhibited by the inhibitory CAR binding to its ligand, e.g., reducing off-target effects.

In some embodiments, the two receptors induce activating and inhibitory signals, respectively, on the cell, such that ligation of the antigen by one of the receptors activates the cell or induces a response, while ligation of the antigen by the other inhibitory receptor induces a signal that suppresses or reduces the response. An example is the combination of an activating CAR and an inhibitory CAR (iCAR). Such a strategy can be used to reduce the possibility of off-target effects, for example, in situations where an activating CAR binds to an antigen expressed in a disease or condition but also expressed on normal cells, and an inhibitory receptor binds to another antigen expressed on normal cells but not on cells with the disease or condition.

In some aspects, the chimeric receptor is or includes an inhibitory CAR (e.g., iCAR) and includes an intracellular component that reduces or suppresses an immune response, e.g., ITAM-promoted and/or costimulatory-promoted responses, in a cell. Exemplary such intracellular signaling components are those found in immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2/4 adenosine receptors, including A2AR. In some aspects, engineered cells include inhibitory CARs that include signaling domains of or derived from such inhibitory molecules, which thus act to reduce cellular responses elicited, e.g., by activating and/or costimulatory CARs.

In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane domain and signaling domain linked to a CD3 (e.g., CD3 zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR includes one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3 zeta, CD28, and 4-1BB.

In some embodiments, the antigen receptor further comprises a marker, and/or the cells expressing the CAR or other antigen receptor further comprise a surrogate marker, such as a cell surface marker, that can be used to confirm transduction or engineering of the cells to express the receptor. In some aspects, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, or epidermal growth factor receptor, such as a truncated form of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, such as a cleavable linker sequence, such as a polynucleotide encoding T2A. For example, the marker and optionally the linker sequence can be any of those disclosed in published International Patent Application No. 2014031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence, such as a T2A cleavable linker sequence.

Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include an amino acid sequence set forth in SEQ ID NO:43 or SEQ ID NO:44, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:43 or 16. Exemplary T2A linker sequences include an amino acid sequence set forth in SEQ ID NO:47 or SEQ ID NO:48, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:47 or 48.

In some embodiments, the marker is a molecule that is not naturally found on T cells or on the surface of T cells, e.g., a cell surface protein, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells are to be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/or has no effect other than being used as a marker for genetic engineering, e.g., to select successfully engineered cells. In another embodiment, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, e.g., a ligand for cells encountered in vivo, e.g., a co-stimulatory or immune checkpoint molecule to enhance and/or attenuate the response of cells when adoptively transferred and encounter the ligand.

In some cases, the CAR is a first generation CAR, a second generation CAR, and/or a third generation CAR. In some aspects, a first generation CAR simply provides a CD3-chain triggering signal upon antigen binding, a second generation CAR provides such a signal as well as a costimulatory signal, which includes an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137, and in some aspects, a third generation CAR includes multiple costimulatory domains from different costimulatory receptors.

For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, scFv, specific for an antigen that includes a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, as described, and an intracellular signaling domain that includes a signaling portion of CD28 or a functional variant thereof, and a signaling portion of CD3 zeta or a functional variant thereof, as described. In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, scFv, specific for an antigen that includes a transmembrane domain that is or contains a transmembrane portion of 4-1BB or a functional variant thereof, as described, and an intracellular signaling domain that includes a signaling portion of CD28 or a functional variant thereof, and a signaling portion of CD3 zeta or a functional variant thereof, as described. In some such embodiments, the receptor further includes a spacer that includes a portion of an Ig molecule, e.g., an Ig hinge, hinge only spacer, e.g., an IgG4 hinge, as described.

In some embodiments, the transmembrane domain of a recombinant receptor, e.g., CAR, is or comprises a transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or a variant thereof, such as a transmembrane domain comprising a sequence of amino acids set forth in SEQ ID NO:95 or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:95; in some embodiments, the transmembrane domain-containing portion of the recombinant receptor is or comprises a transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or a variant thereof, such as a transmembrane domain comprising a sequence of amino acids set forth in SEQ ID NO:95 or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:95; The amino acid sequence set forth in NO:96 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the intracellular signaling component(s) of a recombinant receptor, e.g., a CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, e.g., a domain having an LL→GG substitution at positions 186-187 of the native CD28 protein. For example, the intracellular signaling domain can include an amino acid sequence set forth in SEQ ID NO:97 or SEQ ID NO:98, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:97 or 98. In some embodiments, the intracellular domain comprises the intracellular costimulatory signaling domain of 4-1BB (e.g., Accession No. Q07011.1) or a functional variant or portion thereof, e.g., a sequence of amino acids set forth in SEQ ID NO:99, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:99.

In some embodiments, the intracellular signaling domain of a recombinant receptor, e.g., a CAR, comprises a human CD3 zeta stimulatory signaling domain or a functional variant thereof, e.g., the 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (accession number P20963.2) or the CD3 zeta domain described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993. For example, in some embodiments, the intracellular signaling domain can comprise an amino acid sequence as set forth in SEQ ID NO:97 or SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102.

In some aspects, the spacer contains only the hinge region of an IgG, e.g., an IgG4 or IgG1 hinge only, e.g., the hinge-only spacer shown in SEQ ID NO:81. In another embodiment, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to the CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the CH2 and CH3 domains as shown in SEQ ID NO:84. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the CH3 domain only as shown in SEQ ID NO:83. In some embodiments, the spacer is or contains a glycine-serine rich sequence or another flexible linker, such as a known flexible linker.

For example, in some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, including an scFv, a spacer, e.g., a portion of an immunoglobulin molecule, e.g., a spacer containing a hinge region and/or a constant region of one or more heavy chain molecules, e.g., an Ig-hinge-containing spacer, a transmembrane domain containing all or a portion of a transmembrane domain from CD28, an intracellular signaling domain from CD28, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment, e.g., an scFv, a spacer, e.g., an Ig-hinge-containing spacer, a transmembrane domain from CD28, an intracellular signaling domain from 4-1BB, and a signaling domain from CD3 zeta.

Exemplary surrogate markers can include truncated cell surface polypeptides, e.g., truncated forms that are non-functional and do not transmit or are not capable of transmitting a signal or that do not transmit or are not capable of transmitting a signal normally transmitted by the full-length cell surface polypeptide and/or do not internalize or are not capable of internalizing. Exemplary truncated cell surface polypeptides include truncated growth factor receptors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, an exemplary tEGFR sequence is shown in SEQ ID NO:43 or SEQ ID NO:44), or prostate specific membrane antigen (PSMA), or modified forms thereof. The tEGFR can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which epitope can be used to identify or select cells engineered with the tEGFR construct and the encoded exogenous protein, and/or to exclude or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April;34(4):430-434. In some aspects, the marker, e.g., surrogate marker, comprises all or a portion (e.g., a truncated form) of CD34, all or a portion (e.g., a truncated form) of NGFR, CD19 or a truncated form of CD19, e.g., a truncated non-human CD19, or all or a portion (e.g., a truncated form) of epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP) and yellow fluorescent protein (YFP), and variants thereof, such as species variants, monomeric variants, and codon-optimized and/or high-sensitivity variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted placental alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescence reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

In some embodiments, the marker is a resistance marker or a selection marker. In some embodiments, the resistance marker or selection marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the resistance marker or selection marker is an antibiotic resistance gene. In some embodiments, the resistance marker or selection marker is an antibiotic resistance gene that confers antibiotic resistance to a mammalian cell. In some embodiments, the resistance marker or selection marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or modified versions thereof.

In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A. For example, the marker and optionally the linker sequence can be any of those disclosed in PCT Publication No. 2014031687.

In some embodiments, the nucleic acid molecule encoding such a CAR construct further comprises a sequence encoding a T2A ribosomal skipping element and/or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosomal skipping element as set forth in SEQ ID NO:47 or SEQ ID NO:48 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:47 or SEQ ID NO:48.

In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be generated to express truncated EGFR (EGFRt) as a non-immunogenic selective epitope (e.g., by introduction of a construct encoding CAR and EGFRt separated by a T2A ribosomal switch to express the two proteins from the same construct), which can then be used as a marker to detect such cells (see, e.g., U.S. Pat. No. 8,802,374). In some embodiments, the sequence encodes the tEGFR sequence shown in SEQ ID NO:43 or SEQ ID NO:44, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:43 or SEQ ID NO:44. In some cases, peptides such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), resulting in a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A, e.g., SEQ ID NO:45), equine rhinitis A virus (E2A, e.g., SEQ ID NO:46), Thosea asigna virus (T2A, e.g., SEQ ID NO:47 or 48), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:49 or 50), as described in U.S. Patent Publication No. 20070116690.

A recombinant receptor, such as a CAR, expressed by cells administered to a subject typically recognizes or specifically binds to a molecule expressed in, associated with, and/or specific for the disease or condition being treated or the cells being treated. Upon specific binding to a molecule, such as an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM transduction signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or a cell or tissue associated with the disease or condition.

2. Chimeric Autoantibody Receptor (CAAR)
In some embodiments, the recombinant receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR binds, e.g., specifically binds, or recognizes, an autoantibody. In some embodiments, a cell expressing CAAR, such as a T cell engineered to express CAAR, can be used to bind and kill an autoantibody-expressing cell, rather than bind to a normal antibody-expressing cell. In some embodiments, the CAAR-expressing cell can be used to treat an autoimmune disease associated with the expression of an autoantigen, such as an autoimmune disease. In some embodiments, the CAAR-expressing cell can target B cells that ultimately produce and present the autoantibody on the cell surface, marking these B cells as disease-specific targets for therapeutic intervention. In some embodiments, the CAAR-expressing cell can be used to efficiently target and kill pathogenic B cells in an autoimmune disease by targeting disease-causing B cells using an antigen-specific chimeric autoantibody receptor. In some embodiments, the recombinant receptor is a CAAR, such as any described in US Patent Application Publication No. 2017/0051035.

In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling regions or domains (also interchangeably referred to as cytoplasmic signaling domains or regions). In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of stimulating and/or inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component (e.g., an intracellular signaling domain or region of the CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof), and/or a signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM).

In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The choice of autoantigen can depend on the type of autoantibody being targeted. For example, an autoantigen can be selected because it recognizes an autoantibody on a target cell, e.g., a B cell, that is associated with a particular disease state, e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease includes pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsg1) and Dsg3.

3. T cell receptor (TCR)
In some embodiments, engineered cells, e.g., T cells, are provided that express a T cell receptor (TCR) or an antigen-binding portion thereof that recognizes a peptide epitope or a T cell epitope of a target polypeptide, such as an antigen of a tumor, virus, or autoimmune protein.

In some embodiments, a "T cell receptor" or "TCR" is a molecule that includes variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRα and TCRβ, respectively), or an antigen-binding portion thereof, and is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is of the αβ type. Typically, TCRs that exist in the αβ and γδ types are generally structurally similar, although T cells that express them may have different anatomical locations or functions. TCRs may exist on the surface of a cell or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules.

Unless otherwise indicated, the term "TCR" should be understood to encompass not only complete TCRs, but also antigen-binding portions or fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including αβ or γδ TCRs. In some embodiments, the TCR is an antigen-binding portion of less than the full-length TCR, but which binds to a particular peptide bound to an MHC molecule, such as binding to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR may include only a portion of the structural domain of the full-length or intact TCR, but is capable of binding to a peptide epitope, such as an MHC-peptide complex to which the complete TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR, e.g., the variable α and variable β chains of the TCR, to form a binding site for binding to a particular MHC-peptide complex. Generally, the variable chains of the TCR include the complementarity determining regions involved in recognition of peptides, MHC and/or MHC-peptide complexes.

In some embodiments, the variable domain of a TCR comprises hypervariable loops or complementarity determining regions (CDRs), which are generally the major contributors to antigen recognition and binding capacity and specificity. In some embodiments, one or a combination of CDRs of a TCR form all or substantially all of the antigen binding site of a given TCR molecule. The various CDRs within the variable region of a TCR chain are generally separated by framework regions (FRs), which typically exhibit less variability (variability) between TCR molecules compared to the CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR involved in antigen binding or specificity, or is the most important of the three CDRs of a given TCR variable region for antigen recognition and/or for interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the α chain can interact with the N-terminal portion of a particular antigenic peptide. In some circumstances, CDR1 of the β chain can interact with the C-terminal portion of the peptide. In some circumstances, CDR2 is the primary CDR that contributes most strongly to or is involved in interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the β chain can include an additional hypervariable region (CDR4 or HVR4), which is generally involved in binding superantigens and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, the TCR may also include a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR may possess an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short C-terminal cytoplasmic tail. In some embodiments, the TCR associates with an invariant protein of the CD3 complex, which is involved in mediating signal transduction.

In some embodiments, a TCR chain comprises one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an α chain or a β chain) comprises two immunoglobulin-like domains, e.g., a variable domain (e.g., Vα or Vβ; typically amino acids 1-116 according to the Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)), and a constant domain adjacent to the cell membrane (e.g., an α chain constant domain, or Cα, typically positions 117-259 according to the Kabat numbering, or a β chain constant domain, or _Cβ , typically positions 117-295 according to the Kabat numbering). For example, optionally the extracellular portion of the TCR formed by the two chains comprises two membrane proximal constant domains and two membrane distal variable domains, each of which comprises a CDR. The constant domain of the TCR may comprise a short connecting sequence, in which cysteine residues form disulfide bonds, thereby linking the two chains of the TCR. In some embodiments, the TCR may have an additional cysteine residue in each of the α and β chains, such that the TCR comprises two disulfide bonds in the constant domain.

In some embodiments, the TCR chains include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains include a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3 and its subunits. For example, a TCR that includes a constant domain along with a transmembrane region can anchor the protein to the cell membrane and associate with an invariant subunit of the CD3 signaling apparatus or complex. The intracellular tails of the CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine-based activation motifs, or ITAMs, that are involved in the signaling capacity of the TCR complex.

In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β chains or γ and δ chains), linked, such as by one or more disulfide bonds.

In some embodiments, the TCR can be generated from known TCR sequences, such as sequences of the Vα, β chains, for which substantially full-length coding sequences are readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments, nucleic acid encoding the TCR can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of nucleic acid encoding the TCR within or isolated from one or more given cells, or by synthesis of publicly available TCR DNA sequences.

In some embodiments, the TCR is obtained from a biological source, e.g., from a cell, e.g., a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other published source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, the TCR is a thymic selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR or an antigen-binding portion or fragment thereof can be synthetically generated from knowledge of the sequence of the TCR.

In some embodiments, the TCR is generated from a TCR identified or selected from screening a library of candidate TCRs against a target polypeptide antigen or its target T cell epitope. The TCR library can be generated by amplification of a repertoire of Va and Vβ from T cells isolated from a subject, including cells present in PBMC, spleen or other lymphoid organs. In some cases, T cells can be amplified from tumor infiltrating lymphocytes (TILs). In some embodiments, the TCR library can be generated from CD4+ or CD8+ T cells. In some embodiments, the TCR can be amplified from a source of T cells from a normal or healthy subject, i.e., a normal TCR library. In some embodiments, the TCR can be amplified from a source of T cells from a diseased subject, i.e., a diseased TCR library. In some embodiments, degenerate primers are used to amplify the gene repertoire of Va and Vβ, such as by RT-PCR in a sample, such as T cells obtained from a human. In some embodiments, the scTv library can be assembled from naive Vα and Vβ libraries, where the amplification products are cloned or assembled such that they are separated by a linker. Depending on the subject and the source of the cells, the library can be HLA allele specific. Alternatively, in some embodiments, the TCR library can be generated by mutagenesis or diversification of parent or scaffold TCR molecules. In some aspects, the TCRs are subjected to directed evolution, such as by mutagenesis, of the α or β chain. In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, the selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, such as by screening to assess CTL activity against a peptide. In some aspects, TCRs, such as those present on antigen-specific T cells, can be selected, such as by avidity, e.g., by a particular affinity or avidity for the antigen.

In some embodiments, the TCR or antigen-binding portion thereof is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, e.g., TCRs with higher affinity for a particular MHC-peptide complex. In some embodiments, directed evolution is accomplished by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display approach involves engineering or modifying a known parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to generate mutagenized TCRs in which one or more residues in the CDRs are mutated, and mutants with desired altered properties, such as higher affinity for a desired target antigen, are selected.

In some embodiments, peptides of a target polypeptide for use in producing or generating a TCR of interest are known or can be readily identified. In some embodiments, peptides suitable for use in generating a TCR or antigen-binding portion can be determined based on the presence of HLA-restricted motifs in a target polypeptide of interest, such as a target polypeptide described below. In some embodiments, peptides are identified using available computer prediction models. In some embodiments, to predict MHC class I binding sites, such models include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237, and SYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-93 2007). In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of the Caucasian population and thus represents an appropriate MHC antigen of choice for use in preparing TCRs or other MHC-peptide binding molecules.

Using computational prediction models, HLA-A0201 binding motifs or cleavage sites for the proteasome and immunoproteasome are known. For predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001) and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1):75-93 2007).

In some embodiments, the TCR or antigen-binding portion thereof may be a recombinantly produced native protein or a mutated form thereof in which one or more properties, such as binding characteristics, have been altered. In some embodiments, the TCR may be from one of a variety of animal species, such as human, mouse, rat, or another mammal. The TCR may be in a cell-associated or soluble form. In some embodiments, for purposes of the provided methods, the TCR is in a cell-associated form expressed on the surface of a cell.

In some embodiments, the TCR is a full-chain TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has a structure as described in WO 03/020763, WO 04/033685, or WO 2011/044186.

In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR has the ability to form a TCR complex with CD3. In some embodiments, either the TCR, including the dTCR or the scTCR, can be linked to a signaling domain that results in an active TCR on the surface of the T cell. In some embodiments, the TCR is expressed on the surface of the cell.

In some embodiments, the dTCR contains a first polypeptide in which a TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR α chain constant region extracellular sequence, a second polypeptide in which a TCR β chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR β chain constant region extracellular sequence, and the first and second polypeptides are linked by a disulfide bond. In some embodiments, this disulfide bond can correspond to a native interchain disulfide bond present in a native dimeric αβ TCR. In some embodiments, an interchain disulfide bond is not present in a native TCR. For example, in some embodiments, one or more cysteines can be introduced into the constant region extracellular sequence of the dTCR polypeptide pair. In some cases, native and non-native disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence that anchors it to the membrane.

In some embodiments, the dTCR contains a TCR α chain containing a variable α domain, a constant α domain and a first dimerization motif attached to the C-terminus of the constant α domain, and a TCR β chain containing a variable β domain, a constant β domain and a first dimerization motif attached to the C-terminus of the constant β domain, where the first dimerization motif and the second dimerization motif readily interact to form a covalent bond between the amino acids in the first dimerization motif and the amino acids in the second dimerization motif that bind the TCR α chain and the TCR β chain together.

In some embodiments, the TCR is an scTCR. Typically, the scTCR can be generated using known methods. See, e.g., Soo Hoo, W.F. et al. PNAS (USA) 89, 4759 (1992); Wulfing, C. and Pluckthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); Internationally Published PCT Nos. WO 96/13593, WO 96/18105, WO 99/60120, WO 99/18129, WO 03/020763, 2011/044186, and Schlueter, C.J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains a non-native disulfide interchain bond introduced to enhance TCR chain association (see, e.g., International Publication No. WO03/020763). In some embodiments, the scTCR is a non-disulfide linked truncated TCR with a heterologous leucine zipper fused to its C-terminus to enhance chain association (see, e.g., International Publication No. WO99/60120). In some embodiments, the scTCR contains a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see, e.g., International Publication No. WO99/18129).

In some embodiments, the scTCR comprises a first segment composed of an amino acid sequence corresponding to a TCR α chain variable region, a second segment composed of an amino acid sequence corresponding to a TCR β chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR β chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR comprises a first segment composed of an α chain variable region sequence fused to the N-terminus of an α chain extracellular constant domain sequence, and a second segment composed of a β chain variable region sequence fused to the N-terminus of a β chain extracellular constant sequence and a transmembrane sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR comprises a first segment composed of a TCR β chain variable region sequence fused to the N-terminus of a β chain extracellular constant domain sequence, and a second segment composed of an α chain variable region sequence fused to the N-terminus of an α chain extracellular constant sequence and a transmembrane sequence, and optionally a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

を有する。 In some embodiments, the linker of the scTCR linking the first and second TCR segments can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence may have, for example, the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence in which the amino acids are glycine and serine. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker is long enough to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but is not too long to block or reduce binding of the scTCR to the target ligand. In some embodiments, the linker can comprise 10-45 amino acids or about 10 to about 45 amino acids, such as 10-30 amino acids or 26-41 amino acid residues, such as 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG) ₅ -P- (SEQ ID NO:38), where P is proline, G is glycine, and S is serine.

has.

In some embodiments, the scTCR comprises a covalent disulfide bond linking residues of the immunoglobulin region of the constant domain of the α chain to residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, there are no interchain disulfide bonds in the native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, both native and non-native disulfide bonds may be desirable.

In some embodiments of dTCRs or scTCRs containing an introduced interchain disulfide bond, there are no native disulfide bonds. In some embodiments, one or more native cysteines that form the native interchain disulfide bond are replaced with another residue, such as, for example, serine or alanine. In some embodiments, the introduced disulfide bond can be formed by mutating non-cysteine residues on the first and second segments to cysteines. Exemplary non-native disulfide bonds of TCRs are described in published International PCT Publication No. WO2006/000830.

In some embodiments, the TCR or antigen-binding fragment thereof exhibits an affinity with an equilibrium binding constant for the target antigen that is ^{at or about} 10 to at or about ¹⁰ M, and individual values and ranges therein. In some embodiments, the target antigen is an MHC- ^peptide complex or ligand.

In some embodiments, the nucleic acid or nucleic acids encoding the TCRs, e.g., the α and β chains, may be amplified by PCR, cloning, or another suitable means, and cloned into a suitable expression vector or vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for growth and propagation, or for expression, or both, such as, for example, plasmids and viruses.

In some embodiments, the vector can be a pUC series (Fermentas Life Sciences), a pBluescript series (Stratagene, LaJolla, Calif.), a pET series (Novagen, Madison, Wis.), a pGEX series (Pharmacia Biotech, Uppsala, Sweden) or a pEX series (Clontech, Palo Alto, Calif.) vector. In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. In some embodiments, plant expression vectors can be used, including pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, viral vectors, such as retroviral vectors, are used.

In some embodiments, recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, the vector can contain control sequences, such as transcriptional and translational initiation and termination codons, specific to the type of host (e.g., bacterial, fungal, plant or animal) into which the vector will be introduced, as needed and considering whether the vector is DNA-based or RNA-based. In some embodiments, the vector can contain a native promoter, optionally linked to the nucleotide sequence encoding the TCR or antigen binding portion (or another MHC peptide binding molecule). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as, for example, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus. Other known promoters are also contemplated.

In some embodiments, to generate a vector encoding the TCR, the α and β chains are PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the generated α and β chains are introduced into a retroviral vector, e.g., a lentiviral vector.

B. Nucleic acid, vector and method for genetic engineering In some embodiments, cells, such as T cells, are genetically engineered to express recombinant receptors. In some embodiments, engineering is performed by introducing one or more polynucleotides that code for recombinant receptors or their parts or components. Also provided are polynucleotides that code for recombinant receptors, and vectors or constructs that include such nucleic acids and/or polynucleotides.

In some embodiments, the polynucleotide encoding the recombinant receptor comprises at least one promoter operably linked to control expression of the recombinant receptor. In some examples, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant receptor. In some embodiments, the polynucleotide can include regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host (e.g., bacterial, fungal, plant, or animal) into which the polynucleotide is to be introduced, as appropriate and considering whether the polynucleotide is DNA-based or RNA-based. In some embodiments, the polynucleotide can include regulatory/control elements such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), 2A sequences, and splice acceptors or donors. In some embodiments, the polynucleotide can include a non-native promoter operably linked to the nucleotide sequence encoding the recombinant receptor and/or one or more additional polypeptides. In some embodiments, the promoter is selected from among RNA pol I, pol II, or pol III promoters. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., CMV, SV40 early region, or adenovirus major late promoter). In other embodiments, the promoter is recognized by RNA polymerase III (e.g., U6 or H1 promoter). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus. Other known promoters are also contemplated.

In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, the Simian Virus 40 early promoter (SV40), the Cytomegalovirus immediate early promoter (CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1 alpha promoter (EF1α), the mouse phosphoglycerate kinase 1 promoter (PGK), and the CMV early enhancer-bound chicken β-actin promoter (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises the MND promoter, a synthetic promoter comprising a modified U3 region of MoMuLV LTR with a myeloproliferative sarcoma virus enhancer (see Challita et al. (1995) J. Virol. 69(2):748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters can include, but are not limited to, the human elongation factor 1 alpha (EF1α) promoter or modified forms thereof or the MND promoter.

In another embodiment, the promoter is a regulated promoter (e.g., an inducible promoter). In some embodiments, the promoter is an inducible or repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or is an analog thereof, or can be bound or recognized by a Lac repressor or a tetracycline repressor, or an analog thereof. In some embodiments, the polynucleotide does not comprise a regulatory element, such as a promoter.

In some cases, the nucleic acid sequence encoding the recombinant receptor includes a signal sequence encoding a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide, for example, the exemplary signal peptide of the GMCSFR alpha chain depicted in SEQ ID NO:40, encoded by the nucleotide sequence depicted in SEQ ID NO:41. In some cases, the nucleic acid sequence encoding the recombinant receptor, for example, a chimeric antigen receptor (CAR), includes a signal sequence encoding a signal peptide. Exemplary signal peptides include, but are not limited to, the GMCSFR alpha chain signal peptide depicted in SEQ ID NO:40, encoded by the nucleotide sequence depicted in SEQ ID NO:40, or the CD8 alpha signal peptide depicted in SEQ ID NO:42.

In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding one or more additional polypeptides, such as one or more markers and/or one or more effector molecules. In some embodiments, the one or more markers comprise a transduction marker, a surrogate marker, and/or a resistance or selection marker. For example, among the introduced additional nucleic acid sequences encoding one or more additional polypeptides are those that can improve the efficacy of the treatment, such as by promoting the survival and/or function of the transplanted cells; those that provide genetic markers for the selection and/or evaluation of cells, for example to assess survival or localization in vivo; those that improve safety by rendering cells susceptible to negative selection in vivo, as described, for example, in Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also WO1992008796 and WO1994028143, which describe the use of bifunctional selectable fusion genes derived from the fusion of a dominant positive selectable marker with a negative selectable marker, and U.S. Pat. No. 6,040,177.

In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells into which a polynucleotide, such as a polynucleotide encoding a recombinant receptor, has been introduced. In some embodiments, the transduction marker can indicate or confirm the modification of a cell. In some embodiments, the surrogate marker is a protein that is co-expressed with the recombinant receptor, such as a CAR, on the cell surface. In certain embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to the nucleic acid sequence encoding the marker, optionally separated by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence. Exogenous marker genes may be utilized in some cases in conjunction with engineered cells to allow for detection or selection of the cells, and in some cases may also facilitate cell removal and/or cell suicide.

Exemplary surrogate markers can include truncated forms of cell surface polypeptides, e.g., the truncated forms are non-functional, do not transmit or are unable to transmit and/or are not internalized, a signal or signals normally transmitted by the full-length form of the cell surface polypeptide. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, e.g., truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences shown in SEQ ID NO:43 or 44), or prostate specific membrane antigen (PSMA) or modified forms thereof, e.g., truncated PSMA (tPSMA). In some aspects, tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with the tEGFR construct and the encoded exogenous protein, and/or to eliminate or separate cells expressing the encoded exogenous protein. See U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g., surrogate marker, includes all or a portion (e.g., truncated form) of CD34, NGFR, CD19, or a truncated CD19, e.g., a truncated non-human CD19. Exemplary polypeptides for truncated EGFR (e.g., tEGFR) include the amino acid sequence set forth in SEQ ID NO: 43 or 44, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 43 or 44.

In some embodiments, the marker is or includes a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as superfold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as variants thereof, such as species variants, monomeric variants, codon-optimized, stabilized and/or enhanced variants of fluorescent proteins. In some embodiments, the marker is or includes an enzyme, such as luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof. In some aspects, expression of the enzyme can be detected by addition of a substrate that can be detected upon expression and functional activity of the enzyme.

In some embodiments, the marker is a resistance marker or a selection marker. In some embodiments, the resistance marker or selection marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the resistance marker or selection marker is an antibiotic resistance gene. In some embodiments, the resistance marker or selection marker is an antibiotic resistance gene that confers antibiotic resistance to a mammalian cell. In some embodiments, the resistance marker or selection marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or a modified form thereof.

Any of the recombinant receptors and/or additional polypeptides described herein can be encoded by one or more polynucleotides that include one or more nucleic acid sequences encoding the recombinant receptor in any combination, orientation, or arrangement. For example, one, two, three, or more polynucleotides can encode one, two, three, or more different polypeptides, such as a recombinant receptor or a portion or component thereof, and/or one or more additional polypeptides, such as a marker and/or an effector molecule. In some embodiments, one polynucleotide includes a nucleic acid sequence encoding a recombinant receptor, such as a CAR, or a portion or component thereof, and a nucleic acid sequence encoding one or more additional polypeptides. In some embodiments, one vector or construct includes another vector or construct that includes a nucleic acid sequence encoding a recombinant receptor, such as a CAR, or a portion or component thereof, and a nucleic acid sequence encoding one or more additional polypeptides. In some embodiments, the nucleic acid sequence encoding the recombinant receptor and the nucleic acid sequence encoding the one or more additional polypeptides are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is upstream of the nucleic acid encoding the one or more additional polypeptides. In some embodiments, the nucleic acid encoding the recombinant receptor is downstream of a nucleic acid encoding one or more additional polypeptides.

In certain cases, one polynucleotide includes nucleic acid sequences encoding two or more different polypeptide chains, e.g., a recombinant receptor and one or more additional polypeptides, e.g., a marker and/or an effector molecule. In some embodiments, the nucleic acid sequences encoding the two or more different polypeptide chains, e.g., a recombinant receptor and one or more additional polypeptides, are present in two separate polynucleotides. For example, two separate polynucleotides are provided, each of which can be transferred or introduced separately into a cell for expression in the cell. In some embodiments, the nucleic acid sequence encoding the marker and the nucleic acid sequence encoding the recombinant receptor are present or inserted at different locations in the genome of the cell. In some embodiments, the nucleic acid sequence encoding the marker and the nucleic acid sequence encoding the recombinant receptor are operably linked to two different promoters.

In some embodiments, such as those in which the polynucleotide comprises a first and a second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains can be operably linked to a promoter, which can be the same or different. In some embodiments, the nucleic acid molecule can include a promoter that drives expression of two or more different polypeptide chains. In some embodiments, such a nucleic acid molecule can be multicistronic (bicistronic or tricistronic; see, e.g., U.S. Pat. No. 6,060,273). In some embodiments, the nucleic acid sequence encoding the recombinant receptor and the nucleic acid sequence encoding one or more additional polypeptides are operably linked to the same promoter, optionally separated by an internal ribosome entry site (IRES) or by a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A element. For example, the exemplary marker, and optionally the ribosome skipping sequence, can be any of those disclosed in PCT Publication No. WO2014031687.

In some embodiments, the transcription unit can be engineered as a bicistronic unit that includes an IRES, which allows for co-expression of gene products (e.g., encoding a recombinant receptor and an additional polypeptide) by messages from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of an RNA that contains two or three genes (e.g., encoding a marker and encoding a recombinant receptor) in a single open reading frame (ORF) separated from each other by sequences encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into individual proteins during (in the case of 2A) or after translation. In some cases, peptides such as T2A can cause the ribosome to skip synthesis of a peptide bond at the C-terminus of the 2A element (ribosomal skipping), leading to a separation between that end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). A variety of 2A elements are known. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A, e.g., SEQ ID NO: 45), equine rhinitis virus A (E2A, e.g., SEQ ID NO: 46), Thosea asigna virus (T2A, e.g., SEQ ID NO: 47 or 48), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 49 or 50), as described in U.S. Patent Application Publication No. 20070116690.

In some embodiments, the polynucleotide encoding the recombinant receptor and/or additional polypeptides can be contained in a vector or cloned into one or more vectors. In some embodiments, one or more vectors can be used to transform or transfect a host cell, e.g., a cell for manipulation. Exemplary vectors include vectors designed for introduction, propagation and expansion or for expression, or both, e.g., plasmids and viral vectors. In some aspects, the vector is an expression vector, e.g., a recombinant expression vector. In some embodiments, the recombinant expression vector can be prepared using standard recombinant DNA techniques.

In some embodiments, the vector can be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.) vector. In some cases, bacteriophage vectors, such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, can also be used. In some embodiments, plant expression vectors can be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).

In some embodiments, the vector is a viral vector, e.g., a retroviral vector. In some embodiments, the polynucleotide encoding the recombinant receptor and/or additional polypeptide is introduced into the cell via a retroviral or lentiviral vector, or via a transposon (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010) Molecular Therapy 18:1748-1757; and Hackett et al. (2010) Molecular Therapy 18:674-683).

In some embodiments, the one or more polynucleotides are introduced into the cell using a recombinant infectious viral particle, such as a vector derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the one or more polynucleotides are introduced into the T cell using a recombinant lentiviral or retroviral vector, such as a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557).

In some embodiments, the vector is a retroviral vector. In some aspects, retroviral vectors, such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV), have long terminal repeats (LTRs). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning that they have the ability to infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol, and/or env sequences of the retrovirus. Several illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35 (9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. In some embodiments, a polynucleotide encoding a recombinant receptor and/or one or more additional polypeptides is introduced into a composition containing cultured cells, for example, by retroviral transduction, transfection, or transformation.

In some embodiments, one or more polynucleotides are introduced into the T cells using electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are introduced into the T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material, e.g., polynucleotides and/or vectors, into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)) and other approaches described, e.g., in International Patent Application Publication No. WO2014055668, and U.S. Patent No. 7,446,190.

In some embodiments, one or more polynucleotides or vectors encoding the recombinant receptor and/or additional polypeptides can be introduced into cells, e.g., into T cells, either during or after expansion. This introduction of polynucleotides or vectors can be done, for example, with any suitable retroviral vector. The resulting engineered cells can then be released from the first stimulus (e.g., anti-CD3/anti-CD28 stimulation) and then stimulated in the presence of a second type of stimulus (e.g., via the newly introduced receptor). This second type of stimulus can include antigenic stimulation in the form of a peptide/MHC molecule, a cognate (crosslinking) ligand of the genetically introduced receptor (e.g., the natural antigen and/or ligand of the CAR), or any ligand (such as an antibody) that binds directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). See, for example, Cheadle et al., "Chimeric antigen receptors for T-cell based therapy" Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

In some cases, vectors may be used that do not require the cells, e.g., T cells, to be activated. In some such cases, the cells may be selected and/or transduced prior to activation. Thus, the cells may be manipulated before or after culturing the cells, and in some cases simultaneously with or at least during a portion of the culturing.

V. Compositions, formulations and administration methods Also provided are compositions containing stimulatory cells and selected cells, which are optionally genetically engineered (e.g., engineered antigen receptors), such as CAR or TCR, and compositions containing said cells, including pharmaceutical compositions and formulations.Also provided are methods of using said compositions and their uses, such as in the treatment of diseases, conditions and disorders in which antigens are expressed, or in detection methods, diagnostic methods and prognostic methods.

A. Compositions/Formulations The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some aspects, the choice of carrier is determined in part by the particular cells and/or by the method of administration. Thus, a variety of suitable formulations exist. For example, the pharmaceutical composition may include a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; a sugar such as sucrose, mannitol, trehalose or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and/or a non-ionic surfactant such as polyethylene glycol (PEG).

In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

The formulation or composition may also contain two or more active ingredients useful for the particular indication, disease, or condition being treated with the cells, where each activity does not adversely affect the other, preferably one that has complementary activities. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmacologic active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmacologic acceptable salt. Suitable pharma- ceutically acceptable acid addition salts include those derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, e.g., p-toluenesulfonic acid.

The active ingredient may be encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microparticles, microemulsions, nanoparticles and nanocapsules), or macroemulsions. In certain embodiments, the pharmaceutical compositions are formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or as liposomes. Liposomes may serve to target host cells (e.g., T cells or NK cells) to specific tissues. Many methods are available for preparing liposomes, such as those described in Szoka et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

In some aspects, the pharmaceutical composition may use sustained, delayed, and extended release delivery systems to sensitize the area to be treated and provide sufficient time for the composition to be delivered. Many types of release delivery systems are available and known. Such systems avoid continuous administration of the composition, thereby increasing convenience for the subject and the physician.

In some embodiments, the pharmaceutical composition contains a sufficient amount of cells to treat or prevent a disease or condition, such as a therapeutically or prophylactically effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodic evaluation of the treated subject. In the case of continuous administration over several days or more, depending on the condition, treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and may be determined. The desired dosage may be delivered by a single bolus administration of the composition, by multiple boluses of the composition, or by continuous infusion of the composition.

The cells may be administered using standard administration techniques, formulations, and/or devices. Formulations and devices, e.g., syringes and vials, for the storage and administration of the compositions are provided. Administration of cells can be autologous or xenogeneic. For example, immunoresponsive cells or progenitor cells can be obtained from a subject and administered to the same subject or a different, compatible subject. Peripheral blood derived from immunoresponsive cells or their progeny (e.g., from in vivo, ex vivo, or in vitro sources) is administered via local injection, including catheter administration, systemic injection, regional injection, intravenous injection, or parenteral administration. When administered, therapeutic compositions (e.g., pharmaceutical compositions containing genetically engineered immunoresponsive cells) are typically formulated in a unit dose injectable form (solution, suspension, emulsion).

Formulations include those administered orally, intravenously, intraperitoneally, subcutaneously, pulmonary, transdermal, intramuscular, intranasal, buccal, subcutaneously, or via suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell population is administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

In some embodiments, the compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, may be formulated within an appropriate viscosity range that provides a longer contact period for a particular tissue. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions may be prepared by incorporating the cells in a vehicle, such as by mixing with a suitable carrier, diluent, or excipient, such as, for example, sterile water, saline, glucose, dextrose, and the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances, such as, for example, wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffers, gel-enhancing or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the desired formulation. In some aspects, standard texts may be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules.

Formulations used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

B. Methods of Administration Methods of administering cells, populations and compositions, and the use of such cells, populations and compositions to treat or prevent diseases, conditions and disorders, including cancer, are provided. Methods and uses of cells, populations and compositions, and the use of such cells, populations and compositions to treat or prevent diseases, conditions and disorders, including cancer, are provided. In certain embodiments, the cells, populations and compositions are produced and engineered according to any of the provided methods. In some embodiments, the cells, populations and compositions are administered to a subject or patient with a particular disease or condition to be treated, for example, via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells and compositions prepared by the provided methods, such as the engineered compositions and the completed compositions after incubation and/or other processing steps, are administered to a subject, such as a subject at risk for or at risk for a disease or condition. In some aspects, the method thus treats, for example, alleviates, one or more symptoms of a disease or condition, such as by reducing the tumor burden of a cancer expressing an antigen recognized by the engineered T cells.

Such methods and uses include therapeutic methods and uses involving administration of compositions prepared by the provided methods, e.g., cells and engineered compositions after incubation and/or other processing steps and completed compositions, to a subject having a disease, condition or disorder, e.g., cancer, to effect treatment of the disease or disorder. Uses include the use of the compositions in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods and uses thereby treat a disease or condition or disorder, e.g., a tumor or cancer, in a subject.

Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy is described, for example, in U.S. Patent Application Publication No. 2003/0170238 by Gruenberg et al., U.S. Patent No. 4,690,915 by Rosenberg, and Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

As used herein, a "subject" is a mammal, e.g., a human or another animal, typically a human. In some embodiments, the subject to which the cell, cell population, or composition is administered, e.g., a patient, is a mammal, typically a primate, e.g., a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female, or the subject can be of any suitable age, including an infant, a juvenile subject, an adolescent subject, an adult subject, or a geriatric subject. In some embodiments, the subject is a non-primate mammal, e.g., a rodent.

As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to the completion or partial reversal of, or the reduction of, a disease or condition or disorder, or a symptom, side effect, outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or palliating the disease state, and remission or improving prognosis. The term does not imply complete cure of a disease or complete disappearance of any symptoms or one or more effects of all symptoms or outcomes.

As used herein, "delaying the onset of disease" means to postpone, impede, delay, retard, stabilize, inhibit, and/or prolong the onset of a disease (e.g., cancer). The delay may be of varying lengths of time, which may vary depending on the history of the disease and/or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay may, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastases, may be delayed.

As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

As used herein, to "inhibit" a function or activity is to reduce the function or activity when compared to the same condition otherwise, or alternatively, when compared to another condition, except for the condition or parameter of interest. For example, a cell that inhibits tumor growth reduces the growth rate of a tumor when compared to the growth rate of the tumor in the absence of the cell.

An "effective amount" of an agent, e.g., a pharmaceutical formulation, cell, or composition in the context of administration, refers to an amount effective, at the dosages/administrations required and for the period of time required, to achieve a desired result, e.g., a therapeutic or prophylactic result.

A "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective to achieve a desired therapeutic outcome, e.g., for the treatment of a disease, condition, or disorder, and/or the pharmacokinetic or pharmacodynamic effects of the treatment, at the required dosage/administration and for the required duration. The therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the subject, as well as the population of cells administered. In some embodiments, the methods provided involve administering cells and/or compositions in a therapeutically effective amount, e.g., a therapeutically effective amount.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount will be less than a therapeutically effective amount, since a prophylactic dose is usually used in pre-diseased subjects or subjects at an early stage of disease.

The disease or condition to be treated can be any in which expression of an antigen is associated with and/or involved in the cause of the disease, condition, or disorder, e.g., causes, exacerbates, or is otherwise involved in such disease, condition, or disorder. Exemplary diseases and conditions can include malignancies or cellular transformations (e.g., cancer), autoimmune or inflammatory diseases, or infectious diseases caused, e.g., by bacterial, viral, or other pathogens. Exemplary antigens, including antigens associated with various diseases and conditions that can be treated, are described above. In certain embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease or condition.

Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of cells or compositions containing cells to a subject, tissue, or cell, e.g., having, at risk for, or suspected of having a disease, condition, or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having a particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to a subject, e.g., having or at risk for a disease or condition, to ameliorate one or more symptoms of the disease or condition.

In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous gene transfer, where cells are isolated and/or otherwise prepared from a subject to be treated with cell therapy, e.g., a first subject, or from a sample derived from such a subject. Thus, in some aspects, cells are derived from a subject in need of treatment, e.g., a patient, and after isolation and processing, the cells are administered to the same subject.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by allogeneic gene transfer, where cells are isolated and/or otherwise prepared from a subject other than the subject that will or will ultimately receive cell therapy, such as a first subject. In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. The cells can be administered by any suitable means. Dosage and administration can depend in part on whether administration is brief or chronic. Various administration schedules include, but are not limited to, single or multiple administrations at various time points, boluses, and pulse infusions.

In certain embodiments, the individual populations of cells, or subtypes of cells, are administered to a subject in a range of about 1 million to about 100 billion cells and/or in a cell amount per kilogram of body weight, e.g., about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 700 million cells, about 800 million cells, about 900 million cells, about 1 ... 0 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value in between these ranges and/or per kilogram of body weight. Additionally, dosages may vary depending on the characteristics of the disease or disorder and/or the patient and/or other treatments. In some embodiments, the cells are administered as part of a combination therapy, such as simultaneously with another therapeutic treatment, e.g., an antibody or engineered cell or receptor or an agent such as a cytotoxic drug or therapeutic agent, or sequentially in any order with another therapeutic treatment. In some embodiments, the cells are administered with one or more additional therapeutic agents or linked to another therapeutic treatment, either simultaneously or sequentially in any order. In some situations, the cells are administered with another therapy close enough in time that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, e.g., IL-2, to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

After administration of the cells, the biological activity of the engineered cell population in some embodiments is measured, for example, by any of a number of known methods. Parameters evaluated include specific binding of engineered or natural T cells or other immune cells to an antigen in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, for example, cytotoxicity assays as described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009), and Herman et al. J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and/or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by measuring a clinical outcome, such as reduction in tumor burden or tumor mass.

In certain embodiments, the engineered cells are further modified in a number of ways to increase their therapeutic or prophylactic efficacy. For example, the engineered CAR or engineered TCR expressed by the population can be conjugated to a targeting moiety either directly or indirectly via a linker. The practice of conjugating a compound, such as a CAR or TCR, to a targeting moiety is known in the art. See, for example, Wadwa et al., J. Drug Targeting 3:1 1 1 (1995) and U.S. Patent No. 5,087,616.

VI. Apparatus and Articles of Manufacture In some embodiments, an apparatus or article of manufacture is also provided. To carry out any of the provided methods, an apparatus is provided herein that includes a stationary phase that is an affinity chromatography matrix. In some embodiments, the stationary phase is included in a chromatography column. The arrangement may further include a second stationary phase that is fluidly connected to the first stationary phase. The second stationary phase may be a gel filtration matrix and/or an affinity chromatography matrix, the gel filtration chromatography matrix and/or the affinity chromatography matrix includes a selection reagent, thereby suitable for immobilizing a multimerization reagent on the stationary phase. This type of arrangement can promote sequential selection of target cells (e.g., T cells, CD4, CD3, CD8 T cells), one of which is also suitable for on-column stimulation as described herein.

In some embodiments, the device includes an arrangement that includes a stationary phase that is an affinity chromatography matrix for chromatography (e.g., column chromatography). In some embodiments, the stationary phase has a selection reagent immobilized thereon, e.g., as described herein. In some embodiments, the arrangement includes two of the aforementioned stationary phases for affinity chromatography (e.g., column chromatography) in parallel for selection and on-column stimulation as described herein. In some embodiments, the arrangement includes two of the aforementioned stationary phases for affinity chromatography (e.g., column chromatography) for sequential selection and on-column stimulation as described herein. In some embodiments, the arrangement includes more than two columns that may be arranged to perform parallel and sequential selection, on-column stimulation, or polishing. For example, two columns in parallel may be arranged such that the output of the columns is fed to a column for sequential selection.

In some embodiments, the apparatus contains a stationary phase arrangement (e.g., as described above) and a device (e.g., a centrifugation chamber) for washing and transducing selected cells and stimulator cells collected from the stationary phase.

In some embodiments, the invention further relates to an apparatus for purification (e.g., selection and on-column stimulation) and culturing, e.g., stimulation or expansion of a composition of cells, the apparatus comprising a bioreactor and at least one arrangement of a first or second stationary phase for chromatography as defined above. In some embodiments, an arrangement of a first stationary phase for chromatography and a bioreactor is provided. The bioreactor is suitable for expansion of cells, and the stationary phase is suitable for cell separation and on-column stimulation. For example, the selected cells and the stimulated cells from the stationary phase may be washed, transduced and placed in the bioreactor for expansion. In embodiments, the stationary phase is a gel filtration matrix and/or an affinity chromatography matrix, the gel filtration chromatography matrix and/or the affinity chromatography matrix comprising a selection reagent, the selection reagent comprising a binding site Z1 that specifically binds to a binding partner C1 comprised in the selection substance, and/or the selection reagent comprising a binding site Z2 that specifically binds to a binding partner C2 comprised in the second selection substance. The stationary phase is thereby suitable for immobilization on the first selection agent and/or the second agent, the first binding partner C1 and/or the second binding partner C2. In addition, the bioreactor and the stationary phase are fluidly connected. In some embodiments, the stationary phase may be connected to the bioreactor via a device for washing and transfection steps. This arrangement may be used in continuous expansion. Furthermore, it may be integrated into known cell expansion systems such as the Quantum® Cell Expansion System or the Xuri Cell Expansion System W25. In some embodiments, the bioreactor is connected to a device such as, but not limited to, the Ovizio iLine F (Ovizio Imaging Systems NV/SA, Brussels, Belgium) for monitoring cell health and phenotype (see Section I-E-3a).

In some embodiments, the arrangement further comprises a stationary phase for polishing (see, e.g., Sections I-I). In some embodiments, the stationary phase is an affinity chromatography matrix for chromatography (e.g., column chromatography). In some embodiments, the stationary phase for polishing has a selection reagent, such as those described herein, immobilized thereon to enhance cell selection. In some embodiments, the arrangement further comprises a device (e.g., a centrifugation chamber) for washing, formulating, and filling a suitable container with the therapeutic composition, e.g., as described herein.

In some embodiments, the components of the arrangement are connected by tubing. In some embodiments, the components of the arrangement are connected by welding. In some embodiments, the connections (e.g., tubing and/or welding) between the components of the arrangement are sterile.

In some embodiments, the arrangement is incorporated into a device. The device may further comprise a plurality of arrangements of bioreactors and stationary phases in fluidic connection in series.
The device may comprise a sample inlet fluidly connected to a stationary phase for chromatography (e.g., selection and on-column stimulation). The device may also comprise a sample outlet for purified (e.g., selection) target cells and stimulated target cells, the sample outlet being fluidly connected to the last stationary phase of at least one arrangement of the bioreactor and stationary phase for chromatography. In some embodiments, the device further comprises an outlet, by which the engineered T cells (e.g., therapeutic cell composition) can be loaded into a suitable container. Finally, the device may be designed as a functionally closed system.

VII. DEFINITIONS Unless otherwise defined, all terminology, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and/or ready reference, but the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

As used herein, the singular forms "a," "an," and "the" include the plural of their respective objects unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." Aspects and variations described herein are understood to include "consisting of" and/or "consisting essentially of" aspects and variations.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values within that range. For example, when a range is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the breadth of the range.

As used herein, the term "about" refers to the normal range of error for each stated value. Reference herein to a value or parameter with "about" includes (and describes) aspects directed to that value or parameter itself. For example, a reference to "about X" includes the description of "X."

As used herein, a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence as shown in the sequence listing refers to the nucleotide or amino acid position identified when aligned with the disclosed sequence using a standard alignment algorithm, such as the GAP algorithm, to maximize identity. By aligning the sequences, corresponding residues can be identified, for example, using conserved and identical amino acid residues as a guide. Typically, to identify corresponding positions, amino acid sequences are aligned to obtain the top match (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New. Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Vectors include viral vectors, such as retroviral vectors, e.g., gamma retroviral vectors, and lentiviral vectors.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are also included herein.

As used herein, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence of the particular marker, typically a surface marker, on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, e.g., by staining with and detecting an antibody that specifically binds to the marker, where the staining is detectable by flow cytometry at a level substantially greater than that detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and/or at a level substantially similar to that of cells known to be positive for that marker, and/or at a level substantially greater than that of cells known to be negative for that marker.

As used herein, a statement that a cell or population of cells is "negative" for a particular marker refers to the substantial absence of detectable presence of the particular marker, typically a surface marker, on or in the cells. When referring to a surface marker, the term refers to the absence of surface expression detected by flow cytometry, e.g., by staining with and detecting an antibody that specifically binds to the marker, where the staining is not detected by flow cytometry at a level substantially greater than that detected using an isotype-matched control under otherwise identical conditions using the same procedure, and/or at a level substantially less than that of cells known to be positive for that marker, and/or at a level substantially similar to that of cells known to be negative for that marker.

As used herein, "percent (%) amino acid sequence identity" and "percent identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., an antibody or fragment of interest) that are identical to the amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, not counting conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR, Inc.) software. Appropriate parameters for aligning sequences can be determined, including the algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

Amino acid substitutions involve replacing one amino acid in a polypeptide with another amino acid. Substitutions may be conservative or non-conservative amino acid substitutions. Amino acid substitutions can be introduced into a binding molecule of interest (e.g., an antibody) or into products that are screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Amino acids can generally be divided into groups according to the following common side chain properties:
(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) Basic: His, Lys, Arg;
(5) Residues that affect chain orientation: Gly, Pro;
(6) Aromatics: Trp, Tyr, Phe.

In some embodiments, conservative substitutions include replacing a member of one of these classes with another member of the same class. In some embodiments, non-conservative amino acid substitutions include replacing a member of one of these classes with a member of another class.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, a "subject" is a mammal, such as a human or other animal, typically a human.

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグをさらに含む、態様25の方法。
30. 第1刺激物質および第2刺激物質が、独立して、ビオチン、ストレプトアビジンまたはアビジンに可逆的に結合するビオチン類似体、

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグをさらに含む、態様26の方法。
31. 選択物質が、抗体フラグメント、一価抗体フラグメント、免疫グロブリン様機能を持つタンパク質性結合分子、Igドメインを含有する分子、サイトカイン、ケモカイン、アプタマー、MHC分子、MHC-ペプチド複合体;受容体リガンド;およびその結合性フラグメントからなる群より選択される物質であるか、もしくはそれを含み;ならびに/または
選択物質が抗体フラグメントを含み;
選択物質がFabフラグメントであるか、もしくはそれを含み;
選択物質が、（Fab）₂’フラグメントおよび二価一本鎖Fv（scFv）フラグメントからなる二価抗体フラグメントの群から選択され;
選択物質が、Fabフラグメント、FvフラグメントおよびscFvからなる群より選択される一価抗体フラグメントであり;ならびに/または
選択物質が、アプタマー、リポカリンファミリーのポリペプチドに基づくムテイン、グルボディ、アンキリンスキャフォールドに基づくタンパク質、結晶スキャフォールドに基づくタンパク質、アドネクチンおよびアビマーからなる群より選択される、抗体様結合特性を持つタンパク質性結合分子である、
態様1～30のいずれかの方法。
32. 選択物質が、ビオチン、ストレプトアビジンまたはアビジンに可逆的に結合するビオチン類似体、

からなる群より選択されるストレプトアビジン結合ペプチド、カルモジュリンに可逆的に結合するカルモジュリン結合ペプチド、FLAGペプチドに結合する抗体に可逆的に結合するFLAGペプチド、およびオリゴヒスチジンタグに結合する抗体に可逆的に結合するオリゴヒスチジンタグをさらに含む、態様31の方法。
33. 選択マーカーがT細胞補助受容体であり;
選択マーカーがT細胞抗原受容体複合体のメンバーであるか、もしくはそれを含み、
選択マーカーがCD3鎖であるか、もしくはそれを含み、
選択マーカーがCD3ゼータ鎖であるか、もしくはそれを含み、
選択マーカーがCD8であるか、もしくはそれを含み;
選択マーカーがCD4であるか、もしくはそれを含み、
選択マーカーがCD45RAであるか、もしくはそれを含み;
選択マーカーがCD27であるか、もしくはそれを含み、
選択マーカーがCD28であるか、もしくはそれを含み、および/または
選択マーカーがCCR7であるか、もしくはそれを含む、
態様1～32のいずれかの方法。
34. 選択物質と選択マーカーの間の特異的結合が、T細胞へのシグナルを誘導しないか、または、T細胞への刺激シグナル、活性化シグナルおよび増殖シグナルをいずれも誘導しない、態様1～33のいずれかの方法。
35. 選択物質が抗CD3 Fab、抗CD8 Fabまたは抗CD4 Fabである、態様1～34のいずれかの方法。
36. 刺激試薬が可溶性である、態様2および態様5～35のいずれかの方法。
37. 刺激試薬が、固形支持体、固定相、ビーズ、微粒子、磁気粒子および/もしくはマトリックスではなく、かつそれらに結合も会合もしておらず;ならびに/または
該試薬が、フレキシブルであり、金属コアも磁気コアも含有せず、完全にもしくは主として有機多量体で構成され、形状は球状ではなく、実質的に球状でも均一でもなく、かつ/もしくは剛直ではない、
態様2および態様5～36のいずれかの方法。
38. 刺激試薬が、ストレプトアビジン;アビジン;ビオチン、ビオチン類似体もしくは生物学的に活性なそのフラグメントに可逆的に結合するストレプトアビジンのムテイン;ストレプトアビジン結合ペプチドに可逆的に結合する、アビジンもしくはストレプトアビジンのムテイン;少なくとも2つのキレート基Kを含む試薬であって、該少なくとも2つのキレート基が、遷移金属イオンに結合する能力を有する、試薬;オリゴヒスチジンアフィニティータグに結合する能力を有する物質;グルタチオン-S-トランスフェラーゼに結合する能力を有する物質;カルモジュリンまたはその類似体;カルモジュリン結合ペプチド（CBP）に結合する能力を有する物質;FLAG-ペプチドに結合する能力を有する物質;HAタグに結合する能力を有する物質;マルトース結合タンパク質（MBP）に結合する能力を有する物質;HSVエピトープに結合する能力を有する物質;mycエピトープに結合する能力を有する物質;またはビオチン化担体タンパク質に結合する能力を有する物質であるか、またはそれを含む、態様2および態様5～10、および態様12～37のいずれかの方法。
39. 刺激試薬が、ビオチンもしくは生物学的に活性なフラグメントに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含み、
刺激試薬が、ビオチン類似体もしくは生物学的に活性なフラグメントに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含み、および/あるいは
刺激試薬が、ストレプトアビジン結合ペプチドに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含む、
態様2、5～10および態様12～38のいずれかの方法。
40. 刺激試薬が、複数のストレプトアビジン分子またはストレプトアビジンムテイン分子を含むオリゴマー刺激試薬であり、該オリゴマー粒子試薬のサイズが、i）50nm超の半径、ii）少なくとも5×10⁶g/molの分子量および/または（iii）オリゴマー粒子試薬あたり少なくとも100個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体を含む、態様2、5～10および態様12～39のいずれかの方法。
41. オリゴマー刺激試薬が可溶性である、態様11または態様40の方法。
42. オリゴマー刺激試薬が、固形支持体、固定相、ビーズ、微粒子、磁気粒子および/もしくはマトリックスではなく、かつそれらに結合も会合もしておらず、ならびに/または
該試薬が、フレキシブルであり、金属コアも磁気コアも含有せず、完全にもしくは主として有機多量体、iで構成され、かつ/もしくは剛直ではない、
態様11、態様40または態様41のいずれかの方法。
43. ストレプトアビジン分子またはストレプトアビジンムテイン分子が、ビオチン、ビオチン類似体もしくはストレプトアビジン結合ペプチドに可逆的に結合するか、または可逆的に結合する能力を有する、態様11または態様40～42のいずれかの方法。
44. ストレプトアビジンムテインが、SEQ ID NO:1に示されるアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Va1⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷もしくはlle⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷を含むか、または
ストレプトアビジンムテインが、SEQ ID NO:1に示されるアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Va1⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷を含む、
態様38～43のいずれかの方法。
45. ストレプトアビジン結合ペプチドが、

からなる群より選択される、態様38、39、43または44のいずれかの方法。
46. オリゴマー粒子試薬が、
60nm超、70nm超、80nm超または90nm超の半径
を含む、態様11または40～45のいずれかの方法。
47. オリゴマー粒子試薬が
両端の値を含む50nm～150nm、75nm～125nm、80nm～115nmもしくは90nm～110nmの半径、または
90nm±15nmもしくは95nm±20～25nmの半径
を含む、態様11または態様40～46のいずれかの方法。
48. 前記半径が流体力学的半径である、態様11または40～47のいずれかの方法。
49. オリゴマー粒子試薬が、
少なくとも5×10⁷g/molもしくは少なくとも1×10⁸g/mol、および/または
5×10⁷g/mol～5×10⁸g/mol、1×10⁸g/mol～5×10⁸g/mol、もしくは1×10⁸g/mol～2×10⁸g/mol
の分子量を含む、態様11または40～48のいずれかの方法。
50. オリゴマー粒子試薬が、
少なくとも500個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、少なくとも1,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、少なくとも1,500個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、または少なくとも2,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、および/または
1,000～20,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、1,000～10,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体、または2,000～5,000個のストレプトアビジン四量体もしくはストレプトアビジンムテイン四量体
を含む、態様11または態様40～49のいずれかの方法。
51. 選択物質が固定相に直接的または間接的に結合される、態様1～50のいずれかの方法。
52. 選択物質が、該選択物質が可逆的に結合する選択試薬を介して間接的に固定相に結合される、態様1～51のいずれかの方法。
53. 選択試薬が、ストレプトアビジン;アビジン;ビオチン、ビオチン類似体もしくは生物学的に活性なそのフラグメントに可逆的に結合するストレプトアビジンのムテイン;ストレプトアビジン結合ペプチドに可逆的に結合する、アビジンもしくはストレプトアビジンのムテイン;少なくとも2つのキレート基Kを含む試薬であって、該少なくとも2つのキレート基が、遷移金属イオンに結合する能力を有する、試薬;オリゴヒスチジンアフィニティータグに結合する能力を有する物質;グルタチオン-S-トランスフェラーゼに結合する能力を有する物質;カルモジュリンまたはその類似体;カルモジュリン結合ペプチド（CBP）に結合する能力を有する物質;FLAG-ペプチドに結合する能力を有する物質;HAタグに結合する能力を有する物質;マルトース結合タンパク質（MBP）に結合する能力を有する物質;HSVエピトープに結合する能力を有する物質;mycエピトープに結合する能力を有する物質;またはビオチン化担体タンパク質に結合する能力を有する物質であるか、またはそれを含む、態様51または態様52の方法。
54. 選択試薬が、ビオチンもしくは生物学的に活性なフラグメントに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含み、
刺激試薬が、ビオチン類似体もしくは生物学的に活性なフラグメントに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含み、および/あるいは
刺激試薬が、ストレプトアビジン結合ペプチドに可逆的に結合するストレプトアビジンムテインもしくはアビジンムテインであるか、またはそれを含む、
態様51～53のいずれかの方法。
55. ストレプトアビジン分子またはストレプトアビジンムテイン分子が、ビオチン、ビオチン類似体もしくはストレプトアビジン結合ペプチドに可逆的に結合するか、または可逆的に結合する能力を有する、態様53または態様54の方法。
56. ストレプトアビジンムテインが、SEQ ID NO:1に示されるアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Va1⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷もしくはlle⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷を含むか、または
ストレプトアビジンムテインが、SEQ ID NO:1に示されるアミノ酸の配列におけるストレプトアビジン中の位置を基準として位置44～47に対応する配列位置に、アミノ酸配列Va1⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷を含む、
態様53～55のいずれかの方法。
57. ストレプトアビジン結合ペプチドが、

からなる群より選択される、態様53～54のいずれかの方法。
58. 重力流によって収集することが、固定相からT細胞を溶出させるための競合剤も遊離結合剤も含まない培地を固定相に加えることを含む、態様1～10または態様12～57のいずれかの方法。
59. 前記収集後に、刺激されたT細胞を含む組成物をさらにインキュベートする工程を含む、態様1～10または態様12～58のいずれかの方法。
60. さらなるインキュベーションが、37℃±2℃もしくは約37℃±2℃で実行され;および/またはさらなるインキュベーションが、T細胞においてシグナルを送達する能力を有するさらなる作用物質の存在下で実行される、
態様59の方法。
61. さらなる作用物質が、固定相を洗浄するために使用される培地に含まれる、態様60の方法。
62. さらなる作用物質が、T細胞、CD4＋T細胞および/またはCD8＋T細胞の増殖を増強または誘導する能力を有する、態様60または態様61の方法。
63. さらなる作用物質が、IL-2、IL-15およびIL-7の中から選択されるサイトカインである、態様60～62のいずれかの方法。
64. さらなるインキュベーションが、72時間、48時間以下、24時間以下、または12時間以下の時間にわたって実行される、態様59～63のいずれかの方法。
65. 組成物の刺激されたT細胞に、組換えタンパク質をコードする組換え核酸分子を導入し、それによって、形質導入T細胞を含む組成物を作製する工程をさらに含む、態様1～64のいずれかの方法。
66. 組換えタンパク質が抗原受容体である、態様65の方法。
67. 組換えタンパク質がキメラ抗原受容体である、態様65の方法。
68. キメラ抗原受容体（CAR）が、標的抗原に特異的に結合する細胞外抗原認識ドメインと、ITAMを含む細胞内シグナル伝達ドメインとを含む、態様67の方法。
69. 細胞内シグナル伝達ドメインが、CD3ゼータ（CD3ζ）鎖の細胞内ドメインを含む、態様68の方法。
70. 細胞外ドメインと細胞内シグナル伝達ドメインとを連結する膜貫通ドメインをさらに含む、態様68または態様69の方法。
71. 膜貫通ドメインがCD28の膜貫通部分を含む、態様70の方法。
72. 細胞内シグナル伝達ドメインがT細胞共刺激分子の細胞内シグナル伝達ドメインをさらに含む、態様68～71のいずれかの方法。
73. T細胞共刺激分子が、CD28および41BBからなる群より選択される、態様72の方法。
74. 前記核酸が、組換え抗原受容体をコードする核酸に機能的に連結されたプロモータをさらに含む、態様65～73のいずれかの方法。
75. 組換え核酸の導入が、ウイルス粒子を使った形質導入によって達成される、態様65～74いずれかの方法。
76. ウイルス粒子がレトロウイルスベクター粒子である、態様75の方法。
77. ウイルス粒子がレンチウイルスベクター粒子である、態様75の方法。
78. 形質導入細胞を含む組成物を、ウイルス組込みのための条件下で培養し、それによって、培養T細胞を含む組成物を作製する工程を、さらに含む、態様65～77のいずれかの方法。
79. 形質導入細胞を含む組成物を、T細胞を拡大培養するための条件下で培養する工程をさらに含む、態様75～78のいずれかの方法。
80. 培養する工程が14日以下、12日以下、10日以下、8日以下または6日以下の時間にわたって実行される、態様79の方法。
81. 刺激されたT細胞を含む組成物に競合剤または遊離結合剤を加え、それによって、可逆的結合を破壊する工程を、さらに含む、態様59～64のいずれかの方法。
82. 形質導入T細胞を含む組成物に競合剤または遊離結合剤を加え、それによって、可逆的結合を破壊する工程を、さらに含む、態様65～77のいずれかの方法。
83. 培養T細胞を含む組成物に競合剤または遊離結合剤を加え、それによって、可逆的結合を破壊する工程を、さらに含む、態様78～80のいずれかの方法。
84. 競合剤もしくは遊離結合剤が、T細胞にとって有害ではなく、および/または、該物質の添加が、競合剤も遊離結合剤も使用しない同等もしくは同じ条件下でのT細胞のインキュベーションとの比較で、生残するT細胞のパーセンテージを90％未満、80％未満、70％未満、60％未満もしくは50％未満に低減させることはない、態様81～83のいずれかの方法。
85. 前記破壊が、T細胞における刺激シグナルを終結または軽減させる、態様81～84のいずれかの方法。
86. 競合試薬および遊離結合剤が、独立して、ストレプトアビジン結合分子;ビオチン;D-ビオチン;ビオチン類似体;ストレプトアビジン、または野生型ストレプトアビジンの位置44～47に対応する配列位置にアミノ酸配列Va1⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷もしくはlle⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷を有するストレプトアビジン類似体に特異的に結合するビオチン類似体からなる群からの分子;SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:5およびSEQ ID NO:7のいずれか1つに示される配列を含むか、またはこれからなるペプチドを含み、または
競合試薬および遊離結合剤が、独立して、任意でEDTAまたはEGTAである、金属キレート剤を含む、
態様81～85のいずれかの方法。
87. T細胞が、抗原特異的T細胞もしくはその集団、Tヘルパー細胞もしくはその集団、細胞傷害性T細胞もしくはその集団、メモリーT細胞もしくはその集団、または制御性T細胞もしくはその集団を含む、態様1～77のいずれかの方法。
88. T細胞がCD3＋T細胞を含むか、CD4＋および/またはCD8＋T細胞を含む、態様1～87のいずれかの方法。
89. 固定相がクロマトグラフィーマトリックスであるか、またはそれを含む、態様1～88のいずれかの方法。
90. （a）T細胞表面上の第1分子および第2分子にそれぞれ特異的に結合することによってT細胞を刺激する能力を有する、第1刺激物質および第2刺激物質と、
（b）T細胞上の選択マーカーに特異的に結合することによって固定相上にT細胞を固定化する能力を有する選択物質を含む、固定相と
を含む、T細胞のオンカラム刺激のための製造物品。
91. 固定相が第1刺激物質および第2刺激物質をさらに含む、態様90の製造物品。
92. 第1刺激物質、第2刺激物質および選択物質が、選択試薬を介して間接的に固定相に結合される、態様90または態様91の製造物品。
93. 刺激試薬をさらに含み、第1刺激物質および第2刺激物質が、可逆的に結合されているかまたは可逆的に結合される能力を有する、態様90の製造物品。
94. 選択物質が選択試薬を介して間接的に固定相に結合される、態様90の方法。
95. 固定相が、クロマトグラフィーマトリックスであるかそれを含み、前記製造物品が、該クロマトグラフィーマトリックスの全部または一部を含有する容器をさらに含む、態様90～94のいずれかの製造物品。
96. 態様90～95のいずれかの製造物品を含む、装置。
97. 装置の1つまたは複数の構成要素に流体的に接続された流体入口および/あるいは装置の1つまたは複数の構成要素に流体的に接続された流体出口をさらに含む、態様96の装置。
98. 閉鎖システム内または無菌システム内にある、態様96または態様97のいずれかの装置。
99. 自動で実行されてもよい態様1～89のいずれかの方法において使用するための、態様96～98のいずれかの装置または態様90～95のいずれかの物品。 VIII. Exemplary Embodiments Embodiments provided include the following.
1. A method for on-column stimulation of T cells, comprising the steps of:
(a) incubating a plurality of T cells immobilized on a stationary phase with one or more stimulatory agents to deliver a stimulatory signal in one or more T cells of the plurality of T cells, wherein the stationary phase comprises a selection agent that specifically binds to a selection marker on a surface of the one or more T cells, and wherein specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes the one or more T cells on the stationary phase; and (b) within 24 hours from the start of incubation, collecting the one or more T cells from the stationary phase by gravity flow without adding a competitor or free binding agent to elute the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells.
2. The method of embodiment 1, wherein the stationary phase comprises at least one of one or more stimulatory substances capable of delivering a stimulatory signal in one or more T cells.
3. The method of embodiment 2, wherein the at least one stimulatory substance is a first stimulatory substance, and the stationary phase further comprises one or more of a second stimulatory substance capable of enhancing, attenuating or modifying the stimulatory signal of the first stimulatory substance.
4. The method of embodiment 2, wherein the stimulating agent is a first stimulating agent, and prior to incubation, a stimulating reagent is added to the stationary phase, the stimulating reagent including one or more of a second stimulating agent capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating agent.
5. The method of embodiment 1, wherein prior to incubating, a stimulating reagent comprising at least one of the one or more stimulating substances is added to the stationary phase.
6. The method of embodiment 5, wherein the at least one stimulatory substance is a first stimulatory substance, and the one or more stimulatory substances further comprise one or more second stimulatory substances capable of enhancing, attenuating or modifying the stimulatory signal of the first stimulatory substance.
7. The method of any of embodiments 1-6, wherein at least one of the one or more stimulatory agents, optionally the first stimulatory agent, is capable of delivering a stimulatory signal, the stimulatory signal being a stimulatory signal via the TCR/CD3 complex in the T cell, a CD3-containing complex in the T cell, and/or an ITAM-containing molecule in the T cell.
8. The method of any of embodiments 3, 4, 6, and 7, wherein the second stimulatory agent has the ability to specifically bind to one or more costimulatory molecules on T cells.
9. A method for on-column stimulation of T cells, comprising the steps of:
(a) adding a sample comprising a plurality of T cells to a stationary phase, the stationary phase comprising a selection agent that binds to a selection marker on a surface of one or more of the plurality of T cells, thereby immobilizing the one or more of the plurality of T cells on the stationary phase;
(b) adding a stimulating reagent to the stationary phase, the stimulating reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in one or more of the plurality of T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and (c) within 24 hours from the initiation of incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binding agent to elute the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells.
10. A method for on-column stimulation of T cells, comprising the steps of:
(1) mixing (a) a sample comprising a plurality of T cells with (b) a stationary phase comprising a selection agent capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, wherein specific binding of the selection agent to the selection marker results in immobilization of the plurality of T cells to the stationary phase;
(2) adding a stimulating reagent to the stationary phase, the stimulating reagent comprising one or more stimulatory substances capable of delivering a stimulatory signal in the T cells, thereby initiating incubation of the stimulating reagent with the one or more T cells; and (3) within 24 hours from the initiation of incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binding agent to elute the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells.
11. A method for on-column stimulation of T cells, comprising the steps of:
adding an oligomeric stimulating reagent to a stationary phase comprising the plurality of immobilized T cells, thereby initiating incubation of the stimulating reagent with one or more T cells of the plurality of immobilized T cells;
the stationary phase comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells, and specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes the one or more T cells to the stationary phase;
the oligomeric stimulating reagent comprises (i) a plurality of streptavidin or streptavidin mutein molecules and (ii) one or more stimulatory substances capable of delivering a stimulatory signal in one or more T cells, the size of the oligomeric stimulating reagent being i) a radius of greater than 50 nm, ii) a molecular weight of at least 5×10 ⁶ g/mol, and/or (iii) at least 100 streptavidin or streptavidin mutein tetramers per oligomeric stimulating reagent;
Process.
12. The method of embodiment 11, further comprising the step of collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding a competitor or free binding agent to elute the plurality of T cells from the stationary phase within 24 hours from the start of incubation, thereby producing a composition containing stimulated T cells.
13. The method of any of aspects 1-10 or 12, wherein harvesting one or more of the plurality of T cells from the stationary phase occurs within about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours from the start of incubation.
14. Harvesting one or more of the plurality of T cells from the stationary phase may occur at about 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 19-24, 20-24, or 21-24 hours after the start of incubation. 21-24, 22-24, 23-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 or 2-3 hours.
15. The method of any of aspects 1-10 or 12-14, wherein harvesting one or more of the plurality of T cells from the stationary phase is performed within about 12, 10, 8, 6, 4, or 2 hours from the start of incubation.
16. The method of any of aspects 1-10 or 12-15, wherein harvesting one or more of the plurality of T cells from the stationary phase is performed within 5 hours of initiating incubation.
17. The method of any of aspects 1-10 or 12-16, wherein harvesting one or more of the plurality of T cells from the stationary phase is performed within 4 hours of initiating incubation.
18. The method of any of embodiments 9, 10, and 13-17, wherein the initiation of incubation with the stimulating reagent is performed within or about 10 minutes, within or about 20 minutes, within or about 30 minutes, within or about 45 minutes, within or about 60 minutes, within or about 90 minutes, or within or about 120 minutes after adding or mixing the sample comprising the plurality of T cells to the stationary phase.
19. The method of any of embodiments 9-18, wherein at least one of the one or more stimulatory substances is capable of delivering a stimulatory signal, the stimulatory signal being a stimulatory signal via the TCR/CD3 complex in the T cell, a CD3-containing complex in the T cell, and/or an ITAM-containing molecule in the T cell.
20. The method of embodiment 19, wherein the at least one stimulating substance is a first stimulating substance, and the stimulating reagent further comprises one or more of a second stimulating substance capable of enhancing, attenuating or modifying the stimulating signal of the first stimulating substance.
21. The method of any of embodiments 3, 4, 6, 7, and 20, wherein the second stimulatory agent has the ability to specifically bind to one or more costimulatory molecules on T cells.
22. The method of embodiment 21, wherein the costimulatory molecule is selected from among CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.
23. The method of embodiment 21 or embodiment 22, wherein the second stimulatory agent has the ability to specifically bind to CD28.
24. The method of any of embodiments 3, 4, 6, 7, and 20-23, wherein the first stimulatory agent specifically binds CD3 and the second stimulatory agent specifically binds CD28.
25. The stimulatory agent is or comprises a substance selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and/or the stimulatory agent comprises an antibody fragment;
the stimulator is or comprises a Fab fragment;
the stimulatory agent is selected from the group of bivalent antibody fragments consisting of (Fab) ₂ ' fragments and bivalent single chain Fv (scFv) fragments;
the stimulator is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment and an scFv; and/or the stimulator is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystal scaffold-based protein, an adnectin and an avimer.
The method of any of embodiments 1 to 24.
26. The first stimulatory agent and the second stimulatory agent are, or comprise, independently, an agent selected from the group consisting of an antibody fragment, a monovalent antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, a molecule containing an Ig domain, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex; a receptor ligand; and binding fragments thereof; and/or the first stimulatory agent and the second stimulatory agent comprise, independently, an antibody fragment;
the first stimulatory agent and the second stimulatory agent are, or comprise, independently, a Fab fragment;
the first stimulatory agent and the second stimulatory agent are independently selected from the group of bivalent antibody fragments consisting of (Fab) ₂ ' fragments and bivalent single chain Fv (scFv) fragments;
the first stimulatory agent and the second stimulatory agent are, independently, monovalent antibody fragments selected from the group consisting of Fab fragments, Fv fragments and scFvs; and/or the first stimulatory agent and the second stimulatory agent are, independently, proteinaceous binding molecules with antibody-like binding properties selected from the group consisting of aptamers, muteins based on polypeptides of the lipocalin family, glubodies, ankyrin scaffold-based proteins, crystal scaffold-based proteins, adnectins and avimers.
The method of any of embodiments 3, 4, 6, 7 or 20-24.
27. The method of any of embodiments 3, 4, 6, 7, 20-24, and 26, wherein the first stimulatory reagent is an anti-CD3 Fab and the second stimulatory agent is an anti-CD28 Fab.
28. A method for on-column stimulation of T cells, comprising the steps of:
adding an oligomeric stimulatory reagent capable of delivering a stimulatory signal in a T cell to a stationary phase comprising a plurality of immobilized T cells, thereby initiating incubation of the stimulatory reagent with the one or more T cells,
the stationary phase comprises a selection agent capable of specifically binding to a selection marker on the surface of one or more T cells or a subset thereof, the specific binding of the selection agent to the selection marker expressed by the one or more T cells or a subset thereof resulting in immobilization of the at least a plurality of T cells to the stationary phase, the selection agent being a Fab fragment capable of specifically binding to a selection marker selected from the group consisting of CD3, CD4 and CD8;
The oligomeric stimulating reagent comprises (i) a plurality of streptavidin mutein molecules, (ii) a first stimulating agent which is a Fab fragment capable of specifically binding to CD3 and capable of delivering a stimulatory signal in one or more T cells, and (iii) a second stimulating agent which is a Fab fragment capable of specifically binding to CD28 and capable of enhancing, attenuating or modifying the stimulatory signal, and the size of the oligomeric stimulating reagent comprises: i) a radius of greater than 50 nm; ii) a molecular weight of at least 5 x ¹⁰⁶ g/mol; and/or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric stimulating reagent; and within 24 hours from the start of incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without adding any competitor or free binding agent to elute the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells.
29. A biotin analogue in which the stimulant reversibly binds to biotin, streptavidin or avidin;

26. The method of embodiment 25, further comprising a streptavidin-binding peptide selected from the group consisting of a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.
30. A biotin analogue, wherein the first stimulatory substance and the second stimulatory substance independently reversibly bind to biotin, streptavidin, or avidin;

27. The method of embodiment 26, further comprising a streptavidin binding peptide selected from the group consisting of a calmodulin binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.
31. The selection agent is or comprises a substance selected from the group consisting of antibody fragments, monovalent antibody fragments, proteinaceous binding molecules with immunoglobulin-like functions, molecules containing Ig domains, cytokines, chemokines, aptamers, MHC molecules, MHC-peptide complexes; receptor ligands; and binding fragments thereof; and/or the selection agent comprises an antibody fragment;
the selection agent is or comprises a Fab fragment;
the selected agent is selected from the group of bivalent antibody fragments consisting of (Fab) ₂ ' fragments and bivalent single chain Fv (scFv) fragments;
the selected agent is a monovalent antibody fragment selected from the group consisting of a Fab fragment, an Fv fragment and an scFv; and/or the selected agent is a proteinaceous binding molecule with antibody-like binding properties selected from the group consisting of an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, an ankyrin scaffold-based protein, a crystal scaffold-based protein, an adnectin and an avimer.
The method of any of embodiments 1 to 30.
32. The selective agent is a biotin analogue that reversibly binds to biotin, streptavidin, or avidin;

32. The method of embodiment 31, further comprising a streptavidin-binding peptide selected from the group consisting of a calmodulin-binding peptide that reversibly binds to calmodulin, a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide, and an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag.
33. The selection marker is a T cell coreceptor;
the selectable marker is or comprises a member of the T cell antigen receptor complex;
the selection marker is or comprises the CD3 chain,
the selection marker is or comprises the CD3 zeta chain,
the selection marker is or comprises CD8;
the selection marker is or comprises CD4,
the selection marker is or comprises CD45RA;
the selection marker is or comprises CD27,
The selection marker is or comprises CD28 and/or the selection marker is or comprises CCR7,
The method of any of embodiments 1 to 32.
34. The method of any of embodiments 1 to 33, wherein the specific binding between the selection agent and the selection marker does not induce a signal to the T cell, or does not induce a stimulatory, activating or proliferative signal to the T cell.
35. The method of any of embodiments 1-34, wherein the selection agent is an anti-CD3 Fab, an anti-CD8 Fab, or an anti-CD4 Fab.
36. The method of any of embodiments 2 and 5-35, wherein the stimulating agent is soluble.
37. The stimulation reagent is not, and is not bound to or associated with, a solid support, stationary phase, bead, microparticle, magnetic particle and/or matrix; and/or the reagent is flexible, does not contain a metallic or magnetic core, is composed entirely or primarily of organic polymers, is not spherical, substantially spherical or uniform in shape, and/or is not rigid.
The method of embodiment 2 and any of embodiments 5 to 36.
38. The stimulating reagent is selected from the group consisting of streptavidin; avidin; a mutein of streptavidin that reversibly binds to biotin, a biotin analogue or a biologically active fragment thereof; avidin or a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide; a reagent comprising at least two chelating groups K, the at least two chelating groups being capable of binding to a transition metal ion; a substance capable of binding to an oligohistidine affinity tag; a substance capable of binding to glutathione-S-transferase; a substance capable of binding to a HA tag; a substance capable of binding to a maltose binding protein (MBP); a substance capable of binding to an HSV epitope; a substance capable of binding to a myc epitope; or a substance capable of binding to a biotinylated carrier protein.
39. The stimulating agent is or comprises a streptavidin mutein or avidin mutein that reversibly binds to biotin or a biologically active fragment;
the stimulating reagent is or comprises a streptavidin or avidin mutein that reversibly binds to a biotin analog or biologically active fragment, and/or the stimulating reagent is or comprises a streptavidin or avidin mutein that reversibly binds to a streptavidin binding peptide;
The method of any of embodiments 2, 5-10 and 12-38.
40. The method of any of embodiments 2, 5-10 and 12-39, wherein the stimulation reagent is an oligomeric stimulation reagent comprising a plurality of streptavidin or streptavidin mutein molecules, and the size of the oligomeric particle reagent is: i) a radius of greater than 50 nm; ii) a molecular weight of at least 5×10 ⁶ g/mol; and/or (iii) at least 100 streptavidin tetramers or streptavidin mutein tetramers per oligomeric particle reagent.
41. The method of embodiment 11 or embodiment 40, wherein the oligomeric stimulating reagent is soluble.
42. The oligomeric stimulatory reagent is not, and is not bound to or associated with, a solid support, stationary phase, bead, microparticle, magnetic particle and/or matrix, and/or the reagent is flexible, does not contain a metallic or magnetic core, is composed entirely or primarily of organic polymers, and/or is not rigid.
The method of any of embodiment 11, embodiment 40, or embodiment 41.
43. The method of embodiment 11 or any of embodiments 40 to 42, wherein the streptavidin or streptavidin mutein molecule reversibly binds or has the ability to reversibly bind to biotin, a biotin analogue or a streptavidin-binding peptide.
44. The streptavidin mutein comprises the amino acid sequence Va1 ^{44 -Thr 45 -Ala 46 -Arg 47 or lle 44 -Gly 45 -Ala 46 -Arg 47 at a sequence position corresponding to positions 44 to 47 in streptavidin based on the position in the sequence of amino acids shown in SEQ ID NO:1, or the streptavidin mutein comprises the amino acid sequence Va1 44 -Thr 45 -Ala 46 -Arg 47 at a sequence position corresponding} to positions 44 to 47 in streptavidin based on the position in the sequence of amino acids shown in ^SEQ ID ^NO : ¹ ,
The method of any of embodiments 38 to 43.
45. A streptavidin-binding peptide,

The method of any of embodiments 38, 39, 43, or 44, wherein the method is selected from the group consisting of:
46. The oligomeric particle reagent comprises:
The method of any of embodiments 11 or 40-45, comprising a radius of greater than 60 nm, greater than 70 nm, greater than 80 nm, or greater than 90 nm.
47. The oligomeric particle reagent has a radius of 50 nm to 150 nm, 75 nm to 125 nm, 80 nm to 115 nm, or 90 nm to 110 nm, inclusive; or
The method of embodiment 11 or any of embodiments 40-46, comprising a radius of 90 nm±15 nm or 95 nm±20-25 nm.
48. The method of any of embodiments 11 or 40-47, wherein the radius is a hydrodynamic radius.
49. The oligomeric particle reagent comprises:
at least 5×10 ⁷ g/mol or at least 1×10 ⁸ g/mol, and/or
5×10 ⁷ g/mol to 5×10 ⁸ g/mol, 1×10 ⁸ g/mol to 5×10 ⁸ g/mol, or 1×10 ⁸ g/mol to 2×10 ⁸ g/mol
The method of any of embodiments 11 or 40-48, comprising a molecular weight of
50. An oligomeric particle reagent comprising:
at least 500 streptavidin tetramers or streptavidin mutein tetramers, at least 1,000 streptavidin tetramers or streptavidin mutein tetramers, at least 1,500 streptavidin tetramers or streptavidin mutein tetramers, or at least 2,000 streptavidin tetramers or streptavidin mutein tetramers, and/or
The method of embodiment 11 or any of embodiments 40-49, comprising between 1,000 and 20,000 streptavidin tetramers or streptavidin mutein tetramers, between 1,000 and 10,000 streptavidin tetramers or streptavidin mutein tetramers, or between 2,000 and 5,000 streptavidin tetramers or streptavidin mutein tetramers.
51. The method of any of embodiments 1-50, wherein the selection agent is directly or indirectly bound to the stationary phase.
52. The method of any of embodiments 1-51, wherein the selection agent is indirectly bound to the stationary phase via a selection reagent to which the selection agent reversibly binds.
53. The method of embodiment 51 or embodiment 52, wherein the selection reagent is or comprises streptavidin; avidin; a mutein of streptavidin that binds reversibly to biotin, a biotin analogue or a biologically active fragment thereof; an avidin or a mutein of streptavidin that binds reversibly to a streptavidin-binding peptide; a reagent comprising at least two chelating groups K, said at least two chelating groups being capable of binding to a transition metal ion; a substance capable of binding to an oligohistidine affinity tag; a substance capable of binding to glutathione-S-transferase; calmodulin or an analogue thereof; a substance capable of binding to a calmodulin-binding peptide (CBP); a substance capable of binding to a FLAG-peptide; a substance capable of binding to an HA tag; a substance capable of binding to maltose binding protein (MBP); a substance capable of binding to an HSV epitope; a substance capable of binding to a myc epitope; or a substance capable of binding to a biotinylated carrier protein.
54. The selection agent is or comprises a streptavidin mutein or avidin mutein that reversibly binds biotin or a biologically active fragment;
the stimulating reagent is or comprises a streptavidin or avidin mutein that reversibly binds to a biotin analog or biologically active fragment, and/or the stimulating reagent is or comprises a streptavidin or avidin mutein that reversibly binds to a streptavidin binding peptide;
The method of any of embodiments 51 to 53.
55. The method of embodiment 53 or embodiment 54, wherein the streptavidin molecule or streptavidin mutein molecule reversibly binds or has the ability to reversibly bind to biotin, a biotin analogue or a streptavidin-binding peptide.
56. The streptavidin mutein comprises the amino acid sequence Va1 ^{44 -Thr 45 -Ala 46 -Arg 47 or lle 44 -Gly 45 -Ala 46 -Arg 47 at a sequence position corresponding to positions 44 to 47 in streptavidin based on the position in the sequence of amino acids shown in SEQ ID NO:1, or the streptavidin mutein comprises the amino acid sequence Va1 44 -Thr 45 -Ala 46 -Arg 47 at a sequence position corresponding} to positions 44 to 47 in streptavidin based on the position in the sequence of amino acids shown in ^SEQ ID ^NO : ¹ ,
The method of any of embodiments 53 to 55.
57. A streptavidin-binding peptide,

55. The method of any of embodiments 53-54, wherein the compound is selected from the group consisting of:
58. The method of any of embodiments 1-10 or 12-57, wherein collecting by gravity flow comprises adding medium to the stationary phase that does not contain a competitor or free binder for eluting the T cells from the stationary phase.
59. The method of any of embodiments 1 to 10, or embodiments 12 to 58, comprising, after said collecting, a step of further incubating the composition comprising the stimulated T cells.
60. The further incubation is carried out at or about 37°C ± 2°C; and/or the further incubation is carried out in the presence of a further agent capable of delivering a signal in the T cells.
The method of embodiment 59.
61. The method of embodiment 60, wherein the additional agent is contained in the medium used to wash the stationary phase.
62. The method of embodiment 60 or embodiment 61, wherein the further agent is capable of enhancing or inducing proliferation of T cells, CD4+ T cells and/or CD8+ T cells.
63. The method of any of embodiments 60-62, wherein the additional agent is a cytokine selected from among IL-2, IL-15, and IL-7.
64. The method of any of embodiments 59-63, wherein the further incubation is carried out for a period of 72 hours, 48 hours or less, 24 hours or less, or 12 hours or less.
65. The method of any of embodiments 1-64, further comprising the step of introducing a recombinant nucleic acid molecule encoding a recombinant protein into the stimulated T cells of the composition, thereby producing a composition comprising the transduced T cells.
66. The method of embodiment 65, wherein the recombinant protein is an antigen receptor.
67. The method of embodiment 65, wherein the recombinant protein is a chimeric antigen receptor.
68. The method of embodiment 67, wherein the chimeric antigen receptor (CAR) comprises an extracellular antigen recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM.
69. The method of embodiment 68, wherein the intracellular signaling domain comprises the intracellular domain of the CD3 zeta (CD3ζ) chain.
70. The method of embodiment 68 or embodiment 69, further comprising a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
71. The method of embodiment 70, wherein the transmembrane domain comprises the transmembrane portion of CD28.
72. The method of any of embodiments 68-71, wherein the intracellular signaling domain further comprises an intracellular signaling domain of a T cell costimulatory molecule.
73. The method of embodiment 72, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.
74. The method of any of embodiments 65-73, wherein the nucleic acid further comprises a promoter operably linked to the nucleic acid encoding the recombinant antigen receptor.
75. The method of any of embodiments 65-74, wherein the introduction of the recombinant nucleic acid is achieved by transduction with a viral particle.
76. The method of embodiment 75, wherein the viral particle is a retroviral vector particle.
77. The method of embodiment 75, wherein the viral particle is a lentiviral vector particle.
78. The method of any of embodiments 65-77, further comprising culturing the composition comprising the transduced cells under conditions for viral integration, thereby producing a composition comprising cultured T cells.
79. The method of any of embodiments 75-78, further comprising culturing the composition comprising the transduced cells under conditions for expanding the T cells.
80. The method of embodiment 79, wherein the culturing step is carried out for a period of 14 days or less, 12 days or less, 10 days or less, 8 days or less, or 6 days or less.
81. The method of any of embodiments 59-64, further comprising the step of adding a competitor or free binder to a composition comprising the stimulated T cells, thereby disrupting reversible binding.
82. The method of any of embodiments 65-77, further comprising the step of adding a competitor or free binder to the composition comprising the transduced T cells, thereby disrupting reversible binding.
83. The method of any of embodiments 78-80, further comprising the step of adding a competitor or free binder to the composition comprising the cultured T cells, thereby disrupting the reversible binding.
84. The method of any of embodiments 81-83, wherein the competitor or free binding agent is not detrimental to the T cells and/or addition of the agent does not reduce the percentage of surviving T cells to less than 90%, less than 80%, less than 70%, less than 60%, or less than 50%, compared to incubation of T cells under equivalent or identical conditions without the competitor or free binding agent.
85. The method of any of embodiments 81-84, wherein the disruption terminates or reduces a stimulatory signal in the T cell.
86. The competitive reagent and the free binder independently comprise a molecule from the group consisting of a streptavidin binding molecule; biotin; D-biotin; a biotin analog; a biotin analog that specifically binds to streptavidin or a streptavidin analog having the amino acid sequence ^Va144 - ^Thr45 - ^Ala46 - ^Arg47 or ^lle44 - ^Gly45 - ^Ala46 - ^Arg47 at sequence positions corresponding to positions 44-47 of wild-type streptavidin; a peptide comprising or consisting of a sequence as set forth in any one of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:7; or the competitive reagent and the free binder independently comprise a metal chelator, optionally EDTA or EGTA.
The method of any of embodiments 81 to 85.
87. The method of any of embodiments 1-77, wherein the T cells comprise antigen-specific T cells or a population thereof, T helper cells or a population thereof, cytotoxic T cells or a population thereof, memory T cells or a population thereof, or regulatory T cells or a population thereof.
88. The method of any of embodiments 1-87, wherein the T cells comprise CD3+ T cells, or CD4+ and/or CD8+ T cells.
89. The method of any of embodiments 1-88, wherein the stationary phase is or comprises a chromatographic matrix.
90. (a) a first stimulatory substance and a second stimulatory substance capable of stimulating a T cell by specifically binding to a first molecule and a second molecule, respectively, on the surface of the T cell;
(b) an article of manufacture for on-column stimulation of T cells, comprising: a stationary phase comprising a selection agent capable of immobilizing T cells on the stationary phase by specifically binding to a selection marker on the T cells.
91. The article of manufacture of embodiment 90, wherein the stationary phase further comprises a first stimulant and a second stimulant.
92. The article of manufacture of embodiment 90 or embodiment 91, wherein the first stimulus agent, the second stimulus agent, and the selection agent are indirectly bound to the stationary phase via the selection reagent.
93. The article of manufacture of embodiment 90, further comprising a stimulating reagent, wherein the first stimulating substance and the second stimulating substance are reversibly bound or have the capacity to be reversibly bound.
94. The method of embodiment 90, wherein the selection agent is indirectly bound to the stationary phase via the selection reagent.
95. The article of manufacture of any of embodiments 90-94, wherein the stationary phase is or comprises a chromatography matrix, and the article of manufacture further comprises a vessel containing all or a portion of the chromatography matrix.
96. An apparatus comprising the article of manufacture of any of embodiments 90 to 95.
97. The device of embodiment 96, further comprising a fluid inlet fluidly connected to one or more components of the device and/or a fluid outlet fluidly connected to one or more components of the device.
98. The apparatus of any of aspects 96 or 97, in a closed system or in a sterile system.
99. The apparatus of any of embodiments 96-98 or the article of any of embodiments 90-95, for use in a method of any of embodiments 1-89, which may be performed automatically.

IX. EXAMPLES The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Method for preparing anti-CD3/anti-CD28 Fab-binding oligomeric reagents containing streptavidin muteins
An oligomer reagent was prepared by polymerizing an exemplary streptavidin mutein designated STREP-TACTIN® M2 (a streptavidin homotetramer containing the mutein sequence of amino acids set forth in SEQ ID NO: 6, see, e.g., U.S. Pat. No. 6,103,493 and Voss and Skerra (1997) Protein Eng., 1:975-982 and Argarana et al. (1986) Nucleic Acids Research, 1871-1882). To prepare the streptavidin mutein for oligomerization, a streptavidin mutein containing one or more reactive thiol groups was incubated with a maleimide-activated streptavidin mutein. To prepare the thiolated streptavidin mutein, about 100 mg of streptavidin mutein was thiolated by incubation with 2-iminothiolane hydrochloride in a total volume of 2.6 mL in 100 mM borate buffer at a molar ratio of 1:100 at a pH of about 8.5 and 24° C. for 1 hour. For the activation reaction, about 400 mg of streptavidin mutein was incubated with succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH) in a total volume of about 10.4 mL in sodium phosphate buffer at a molar ratio of 1:2 at a pH of about 7.2 and 24° C. for 1 hour. The thiolation and activation reactions were adjusted to start at approximately the same time, and the duration of the reaction was controlled.

After the reaction, 2-iminothiolane hydrochloride and SMPH were removed from the samples by performing gel filtration of the samples individually using PD-10 desalting columns (GE Healthcare). For each 2.5 mL sample volume, a 1 mL PD-10 column was equilibrated and loaded with thiolated streptavidin or maleimide mutein streptavidin, and elution was performed by adding 3.5 mL of coupling buffer (100 mM _NaH2PO4 , 150 mM NaCl, 5 mM EDTA, pH 7.2). Gel filtration of maleimide mutein streptavidin was performed on four columns occupying >10 mL volumes, and the eluates were pooled. The timing of the activation and thiolation reactions and the timing between the end of the activation and thiolation reactions and the start of the oligomerization reaction were carefully controlled. In general, less than 10 minutes elapsed between the start of gel filtration, i.e., the end of the activation and thiolation reactions and the start of the oligomerization reaction.

The maleimide streptavidin mutein sample and the thiolated streptavidin mutein sample were then combined to a total volume of approximately 17.5 mL for oligomerization and incubated at pH 7.2, 24°C, and stirring at approximately 600 rpm for 1 h. Four times as much streptavidin mutein as in the case of 2-iminothiolane hydrochloride was incubated with SMPH, so the molar ratio of thiolated streptavidin mutein to maleimide streptavidin mutein during the oligomerization reaction was 1:4. After the reaction, the remaining SH groups of the oligomerized streptavidin mutein reagent were saturated by incubating with N-ethylmaleimide (NEM) at 24°C for 15 min with stirring (approximately 600 rpm), followed by an additional 16-20 h incubation at 4°C.

After incubation with NEM, the samples containing the oligomerized streptavidin muteins were centrifuged and the supernatant was filtered through a 0.45 μm membrane (Millex-HP 0.45 μm, Merck Millopore). The filtered solution was then loaded onto a column (Sephacryl S-300 HR HiPrep 26/60, GE Healthcare) and subjected to size-exclusion chromatography (SEC) using an AKTA Explorer chromatography system (GE Healthcare). Fractions with mAU (milli absorbance units) > 1500 mAU were pooled.

Pooled samples containing oligomeric streptavidin muteins were treated with 100 mM hydroxylamine, pH 6.35, for 15 min at room temperature. To remove hydroxylamine after treatment, samples were loaded onto PD10 columns (2.5 mL per column) and eluted with 3.5 mL of buffer, pH 7.2, containing 100 mM NaH2PO4, 140 mM NaCl, 1 mM EDTA. The PD10 eluates were pooled and sterile filtered through a 0.45 μm filter followed by a 0.22 μm filter, after which the samples were frozen and stored at −80°C. Prior to freezing, the final concentration of oligomeric streptavidin mutein reagent was measured and the size of the oligomeric streptavidin mutein reagent was measured by dynamic light scattering (DLS).

To evaluate the consistency of the oligomerization process, ten oligomeric streptavidin mutein reagents were prepared from five different streptavidin mutein (SAM) lots using the method described above. The average size, yield (measured by measuring absorbance at 280 nm, no baseline correction) and activity (biotin binding) of each oligomer were evaluated. The results are shown in Table E1. The results show that the resulting oligomeric streptavidin mutein reagents were consistent in these parameters, with an average radius of 97 nm ± 10 nm and biotin binding of 40 nmol/mg ± 3 nmol/mg.

Table E1. Comparison of oligomerized STREP-TACTIN from different batches

The average molecular weight (MW) of the three oligomeric streptavidin mutein reagents produced as above was measured by asymmetric flow field separation (AF4) performed using HPLC systems (AGILENT 1100 and Wyatt ECLIPSE DUALTEC) with UV detection (Agilent UV detector coupled to MALLS DAWN HELEOS (Wyatt)). AF4 measurements allowed the calculation of the average number of streptavidin mutein tetramers in each oligomeric reagent, assuming an average molecular weight of 52,500 g/mol (52.5 kDa) for the streptavidin mutein tetramers (Table E2).

Table E2: Size and molecular weight of oligomeric streptavidin mutein reagents

を有する2つのストレプトアビジン結合分子の連続配置を含有するストレプトアビジンペプチド結合配列に個々に融着した。ペプチド標識されたFab断片を組換え的に製造した（国際特許出願公開番号WO2013/011011およびWO2013/124474を参照）。 The stimulators (anti-CD3 and anti-CD28 Fab fragments) were multimerized by reversible binding to the oligomeric streptavidin mutein reagents generated as described above. The anti-CD3 and anti-CD28 Fab fragments were reversibly bound to the streptavidin mutein oligomers via streptavidin peptide-binding partners fused to each Fab fragment. The anti-CD3 Fab fragment was derived from a CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™; see also U.S. Pat. No. 4,361,549) and contained the heavy and light chain variable domains of the anti-CD3 antibody OKT3 described in Arakawa et al J. Biochem. 120, 657-662 (1996). These sequences are set forth in SEQ ID NOs: 31 and 32, respectively. The anti-CD28 Fab fragment was derived from the antibody CD28.3 (deposited as a synthetic single chain Fv construct under GenBank accession number AF451974.1; see also Vanhove et al., BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570) and contained the heavy and light chain variable domains of the anti-CD28 antibody CD28.3 as set forth in SEQ ID NOs: 33 and 34, respectively. The Fab fragments were synthesized by cloning the heavy chain carboxy terminus of the antibody CD28.3 to the amino acid sequence

The peptide-tagged Fab fragments were recombinantly produced (see International Patent Application Publication Nos. WO2013/011011 and WO2013/124474).

To perform reversible binding, peptide-labeled anti-CD3 and anti-CD28 Fab fragments were mixed with the oligomeric streptavidin mutein reagent at about room temperature, thereby reversibly binding to the reagent via the interaction between the Twin-Strep-tag on the Fab fragment, which is the binding partner that can reversibly bind to the binding site on the reagent. In some cases, the peptide-labeled Fab fragments were premixed before immobilization on the oligomeric streptavidin mutein reagent, which can result in a more uniform distribution of the various Fab molecules in some instances. The binding of peptide-labeled anti-CD3 and anti-CD28 to the oligomeric streptavidin mutein reagent can be cleaved or eliminated by the addition of D-biotin. D-biotin competes with the Strep-tag on the agent for binding to the binding partner on the streptavidin mutein, thereby cleaving the binding.

Example 2: Activity evaluation of oligomerized anti-CD3 and anti-CD28 Fab fragments reversibly bound to streptavidin mutein oligomers Anti-CD3 and anti-CD28 Fab fragments reversibly bound to various oligomeric streptavidin reagents from each of the batches listed in Table E1 by the process described in Example 1 were evaluated for their ability to stimulate T cells. These oligomeric streptavidin reagents had a mean radius of about 95 nm. Metabolic activity of cells was evaluated as an indicator of cell proliferation by colorimetric monitoring of the cleavage of the stable tetrazolium salt WST-1 to a soluble formazan dye complex.

T cells from three different donors were incubated with anti-CD3/anti-CD28 multimerized Fab fragments reversibly bound to an oligomeric streptavidin reagent. Cells were also incubated with control oligomeric reagents with a mean radius of 101 (internal reference) or 36 nm, also reversibly bound to anti-CD3/anti-CD28 Fab fragments.

After incubation, the level of metabolic activity was assessed by applying WST-1 reagent to the cells and measuring absorbance at 450 nm as readout. Results were normalized to the number of cells in the culture being assayed and expressed as a ratio of WST-1 per cell number.

As shown in Figure 2B, the mean WST-1 activity of T cells stimulated with each of the tested reagents was comparable. Moreover, the degree of stimulation was similar for all tested reagents and comparable to similarly sized internal reference reagents (typically differing within ±2 standard deviations). Figure 2A shows the WST-1 activity plotted as a separate data point for each reagent. Figures 2A and 2B show that the T cell stimulation observed by WST-1 activity was lower when anti-CD3/anti-CD28 Fab multimerized onto the smaller 36 nm oligomeric streptavidin mutein reagent backbone was used.

Example 3: Selection and Stimulation of T Cells by Column Chromatography Studies were performed to enrich for T cells by column-based affinity chromatography with on-column stimulation in the presence of anti-CD3/anti-CD28 oligomer stimulators.

In this study, Sephadex G50 (Sigma) was used as the stationary phase and was covalently coupled to STREP-TACTIN® M2 (SEQ ID NO:6) using cyanogen bromide (CNBr)-activated resin. A 50% suspension of Sephadex G50 contained approximately 70 μg of covalently coupled 7 Strep-tactin® per mL of bead suspension. After immobilization of STREP-TACTIN® onto the stationary phase, 2 mL of the Sephadex G50 suspension containing Strep-tactin® was incubated with 10 μg of selection agent specific for T cell surface selection markers for 20 min at 4°C to allow binding of Fab fragments to the immobilized Strep-tactin® reagent. The suspension was then packed into a plastic mini-column with a 90-micrometer frit at the bottom. The column was equilibrated with PBS (phosphate buffered saline) containing 0.5% bovine serum albumin (PBSA buffer) to obtain a bed volume of 1 mL.

In these studies, experiments were performed using either anti-CD3-binding Fab fragments, anti-CD4-binding Fab fragments or anti-CD8-binding Fab fragments as the selection agents. The CD3-binding Fab fragments were derived from a CD3-binding monoclonal antibody produced by the hybridoma cell line OKT3 (ATCC® CRL-8001™, see also U.S. Pat. No. 4,361,549) and contained the heavy chain variable domain (SEQ ID NO:31) and light chain variable domain (SEQ ID NO:32) of the anti-CD3 antibody OKT3 described by Arakawa et al. J. Biochem. 120, 657-662 (1996). The CD4-binding Fab fragment was obtained from m13B8.2 (e.g., U.S. Pat. No. 7,482,000) and contained the heavy chain variable domain (SEQ ID NO:29) and light chain variable domain (SEQ ID NO:30) of the anti-CD4 antibody m13B8.2. The CD8-binding Fab fragment was obtained from OKT8 (e.g., ATCC CRL-8014) and contained the heavy chain variable domain (SEQ ID NO:9) and light chain variable domain (SEQ ID NO:10) of the anti-CD8 antibody OKT8. The heavy chain of each Fab fragment was fused at the carboxy terminus to Twin Strep-Tag® (SEQ ID NO:16), which contains a consecutive sequence of two streptavidin binding modules.

Apheresis samples from human donors were loaded onto the affinity column and two washing steps were performed. After more than 30 minutes of sample loading time, 40 μg of multimerized anti-CD3 anti-CD28 Fab fragments reversibly bound to oligomeric streptavidin mutein reagent (anti-CD3/anti-CD28 oligomer reagent) produced as described in Example 1 were loaded onto the column in 3 mL of serum-free basal medium containing glutamine and recombinant IL-2, IL-15 and IL-7 and incubated at 37°C. After about 24 hours, the cells were collected from the column in a single step by passing 80 mL of serum-free basal medium containing glutamine and recombinant IL-2, IL-15 and IL-7 through the column without adding any additional competitor to disrupt the binding. As a control, apheresis samples were loaded onto an anti-CD3 affinity column without incubation in the presence of anti-CD3/anti-CD28 oligomer stimulating reagents. For the control condition, D-biotin was added after 24 hours to disrupt the binding between the Twin Strep-Tag® and STREP-TACTIN® of the anti-CD3 Fab, and released cells were collected by gravity flow.

Approximately 24 hours after the start of stimulation with anti-CD3/anti-CD28 oligomer reagents, harvested cells were analyzed for surface expression of selection markers. As shown in Figure 3, downregulation of CD3, CD4, and CD8 was observed 24 hours after on-column selection with the respective selection agents specific for the selection markers and incubation with stimulation reagents. In contrast, no receptor downregulation of the selection markers was observed for the control condition in which cells were not incubated with oligomer stimulation reagents. These results are consistent with the observation that stimulation of T cells with anti-CD3/anti-CD8 stimulation reagents resulted in downregulation of receptor surface expression, thereby allowing cells to spontaneously detach from the column without the need for adding competing reagents.

A similar study was performed in which T cells were selected on an anti-CD3STREP-TACTIN® affinity column as described above and subjected to on-column stimulation with anti-CD3/anti-CD28 oligomer stimulating reagent. Cells were collected by gravity flow at various time points after addition of oligomer stimulating reagent. Collected cells were immediately stained with antibodies against alpha-beta TCR chains and monitored by flow cytometry to detect surface expression of CD3. Figure 4 illustrates exemplary kinetics of downregulation and re-expression of CD3/TCR complexes after initiation of stimulation in the presence of anti-CD3/anti-CD28 oligomer stimulating reagent. As shown, rapid downregulation of surface expression of CD3/TCR complexes was observed during the first 24 hours of stimulation, with the greatest decrease in surface expression of CD3/TCR complexes observed only after 12 hours. Incubation in the presence of oligomeric stimulating reagent for longer than 36 hours resulted in re-expression of surface CD3/TCR complexes, with maximum CD3/TCR complex surface re-expression achieved within about 72 hours after initiation of stimulation with stimulating reagent. These results support that substantial on-column stimulation-mediated spontaneous T cell detachment can occur within 4-6 hours after initiation of on-column incubation with stimulating reagent, with maximum release occurring at or about 12 hours.

Cells that spontaneously detached approximately 24 hours after addition of anti-CD3/anti-CD28 oligomer stimulating reagent were cultured at 37°C in serum-free basal medium containing glutamine and recombinant IL-2, IL-15, and IL-7 for up to 9 days. The oligomer stimulating reagent was not removed during incubation, but was diluted as the cells continued to proliferate during the incubation. Cells were monitored for size and expression of CD3 and activation markers CD69 and CD25 at 24 hours and 5 days during the subsequent incubation. As shown in Figure 5A, further incubation for up to 5 days resulted in re-expression of CD3 after the initial downregulation of CD3 observed at 24 hours. In addition, an increase in cell size and surface expression of activation markers CD25 and CD69 was also observed at day 5 compared to the 24 hours. Assessment of cell number and fold expansion after subsequent incubation for up to 9 days indicated that spontaneously detached cells demonstrated high proliferation capacity (Figure 5B). These results are consistent with the observation that T cells that have undergone on-column selection and short-term stimulation can be further incubated to achieve the desired stimulation, activation and/or proliferation.

These results support the use of on-column selection and stimulation as a rapid means (e.g., less than 24 hours) of isolating and stimulating target cells prior to or in conjunction with downstream processes (e.g., transduction) to generate engineered T cell compositions.

Example 4: Evaluation of T cell phenotype and function in engineered T cells generated in the presence of small molecule mTOR kinase inhibitors CD4+ and CD8+ T cells were isolated by immunoaffinity-based enrichment from leukapheresis samples from human donor subjects. On day 1, isolated CD4+ and CD8+ T cells were mixed 1:1 and stimulated with anti-CD3/anti-CD28 oligomer stimulatory reagents generated as described in Example 1 in serum-free medium supplemented with recombinant IL-2 (100 IU/mL), recombinant IL-7 (600 IU/mL), and recombinant IL-15 (100 IU/mL) with or without 1 μM 2-(3-hydroxyphenyl)-9-(2-isopropylphenyl)-8-oxo-8,9-dihydro-7H-purine-6-carboxamide (compound 63). T cells cultured with or without compound 63 were incubated overnight (about 24 hours) at 37°C and then transduced with a lentiviral vector encoding an anti-CD19 CAR in serum-free medium with or without compound 63 (1 mM). The CAR contained an scFv antigen-binding domain specific for CD19 (derived from FMC63), a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and an intracellular signaling domain derived from CD3-zeta. After transduction, the cells were incubated in serum-free medium without recombinant cytokines (basal medium) with or without compound 63 (1 mM) and allowed to incubate at about 37.0°C in an incubator for up to 96 hours (until day 5 of the process) after the start of stimulation with anti-CD3/anti-CD28 oligomer stimulating reagent. Approximately 24 hours after the start of incubation (day 3 of the process), 1 mM biotin was added. After incubation, CD4+ and CD8+ T cells from each donor were harvested, formulated, and cryofrozen.

Cryofrozen engineered CD4+ and CD8+ T cells were thawed and T cells were assessed for intracellular S6 phosphorylation, markers of ribosomal proteins and mTOR inhibition, and co-stained for surface expression of CD4 or CD8, and CCR7 and CD45RA as markers of memory subsets. pS6 expression in viable CD8+ T cells by memory subsets was indicated by expression of CCR7 and CD45RA as shown in Figure 6A. As shown, incubation with Compound 63 reduced the mean fluorescence intensity of stimulated memory T cell subsets, Temra, Tem, and Tcm cells, indicating inhibition of mTOR, while having no significant effect on the percentage or total number of viable cells over time. The mean fluorescence intensity (MFI) of PS6 in CD8+ T cells is shown in Figure 6B. As shown in Figures 6C and 6D, the presence of Compound 63 did not affect the percent viable cells and total viable cells, respectively, in the resulting compositions.

The thawed cells generated by the described process were evaluated for markers of apoptosis (e.g., percentage of caspase-positive CAR-T cells), phenotypic profile, and ability to produce intracellular cytokines after stimulation with PMA/ionomycin and Golgi inhibitors. As shown in Figure 7A, T cells from samples incubated with Compound 63 exhibited lower intracellular caspase expression than those not incubated with Compound 63, indicating that the presence of Compound 63 during the process of generating engineered cells improves the overall cellular health of the T cell composition. The thawed cells were also stained for surface expression of CD27 and CCR7 by flow cytometry. As shown in Figure 7B (CD8+ T cells) and Figure 7D (CD4+ T cells), incubation with Compound 63 did not substantially alter the phenotypic subset profile of the cells as assessed by expression of CD27 and/or CCR7. The functional activity of CD4+ and CD8+ T cells generated in the presence of Compound 63 was substantially improved in both engineered CD8+ T cells (FIG. 7C) and CD4+ T cells (FIG. 7E) previously generated in the presence of Compound 63, as evidenced by the levels of the intracellular cytokines IL2, IFNg, or TNF.
To further evaluate the functional activity of the cells, the generated T cell composition was stimulated chronically for 12 days with beads conjugated with anti-idiotypic (ID) antibodies against the anti-CD19 CAR, and cell proliferation and survival were monitored (Figure 7F, left panel), and total expansion metrics were calculated by area under the curve (Figure 7F, right panel). As shown, stimulation of cells in the absence of Compound 63 resulted in a decrease in cell proliferation over time and a loss of persistent function after chronic stimulation, consistent with chronic stimulation of the CAR. These results indicate that improved T cell function was observed after chronic CAR-specific stimulation of engineered cells pre-generated in the presence of Compound 63.

Example 5: Evaluation of phenotypic and functional properties of engineered T cells using an integrated selection and on-column stimulation process A process that integrated the selection and stimulation steps in a chromatography column (on-column selection and stimulation) was carried out essentially as described in Example 3, but on a larger scale. The on-column selection and stimulation process was compared to an alternative process that was substantially similar but in which the stimulation step was carried out in solution separate from the selection step.

A. On-column selection and stimulation
On day 0, apheresis samples from human donors were loaded onto an affinity column containing a Sephadex G50 (Sigma) stationary phase covalently coupled to StrepTactin® (SEQ ID NO:6) using cyanogen bromide (CNBr)-activated resin. 20 mL of stationary phase had a capacity of accommodating up to 2 billion ± 500 million cells. The selection material, an anti-CD3 binding Fab fragment as described in Example 3, was immobilized on the stationary phase via a heavy chain carboxy-terminus fused streptavidin binding peptide (Twin Strep-Tag®; SEQ ID NO:16) capable of binding to StrepTactin.

After about 60 minutes from the sample loading time, multimerized anti-CD3 anti-CD28 Fab fragments reversibly bound to oligomeric streptavidin mutein reagent (anti-CD3/anti-CD28 oligomer reagent) produced as described in Example 1 were loaded onto the column at a fixed dose of 0.2-0.3x (1-2 μg/million cells) in serum-free medium containing recombinant IL-2 (e.g., 100 IU/mL), IL-15 (e.g., 100 IU/mL) and IL-7 (e.g., 600 IU/mL) and incubated in the column at 37°C for about 4.5 hours. During incubation, stimulation with anti-CD3/anti-CD28 oligomer reagent resulted in the detachment or release of immobilized cells from the selection material on the column. The released cells were eluted from the column by gravity flow with the same serum-free medium. The medium did not contain biotin or any competing substances that would disrupt the binding between the StrepTactin® on the stationary phase and the streptavidin-binding peptide fused to the anti-CD3 antibody used to immobilize the cells on the stationary phase of the column.

The released and collected cells were then transduced to express a chimeric antigen receptor (CAR) by incubation in the same serum-free medium for 1 hour with a lentiviral vector encoding an exemplary anti-CD19 CAR. The exemplary CAR contained an anti-CD19 scFv from mouse antibody FMC63, an immunoglobulin spacer, a transmembrane domain from CD28, a costimulatory region from 4-1BB, and a CD3-zeta intracellular signaling domain. For transduction, the culture volume was adjusted to 1 x ¹⁰⁶ cells/mL.

The transduced cells were washed and then further incubated at 37°C. Approximately 48 hours after the initiation of on-column stimulation with the anti-CD3/anti-CD28 oligomer reagent, 1.0 mM D-biotin was added and mixed with the cells to dissociate the anti-CD3 Fab and anti-CD28 Fab from the soluble oligomeric streptavidin mutein reagent.

After addition of biotin, the cells were further incubated at 37°C for about an additional 24 hours. The cells were then divided into two subsets. In the first subset, the cells were directly formulated with cryoprotectant. In the second subset, the volume of the cells was adjusted to ^0.5x106 cells/mL in serum-free medium containing twice the concentrations of IL-2, IL-7 and IL-15 used during the incubation and transduction steps. This second subset of cells was further incubated for expansion by culturing for an additional 5 days at 37°C in static culture with medium changes, and then formulated with cryoprotectant.

B. Substitution Process: Selection and Stimulus Separation (in Solution)
The alternative process generally proceeded as described above, but the steps for selection and stimulation were not integrated in the column. Apheresis samples from the same human donors were loaded onto an affinity column containing the anti-CD3 selection reagent described above for the selection of CD3+ T cells. To elute the selected cells, 1.0 mM D-biotin was added to the column and the eluted cells were collected. D-biotin acted as a competitor, disrupting the binding of the streptavidin-binding peptide fused to the anti-CD3 Fab with StrepTactin® on the stationary phase, releasing the cells and anti-CD3 Fab from the column.

On day 0 of the alternative process, the selected cells were washed, diluted to 1×10 ⁶ /mL, and stimulated by incubation with a fixed dose (0.3×, approximately 2 μg/million cells) of anti-CD3/anti-CD28 Fab conjugate oligomeric streptavidin mutein reagent produced as described in Example 1. Stimulation was performed for approximately 18-30 hours (24±6 hours) in serum-free medium containing recombinant IL-2 (e.g., 100 IU/mL), recombinant IL-7 (e.g., 600 IU/mL), and recombinant IL-15 (e.g., 100 IU/mL).

After stimulation, cells were transduced with the lentiviral vector encoding the same exemplary anti-CD19 CAR described above by spinoculation for 30 minutes in the same serum-free medium.

After spinoculation, the cells were washed and then further incubated at 37°C. Approximately 48 ± 6 hours after the start of stimulation with the anti-CD3/anti-CD28 oligomer reagent, 1.0 mM D-biotin was added and mixed with the cells to dissociate the anti-CD3 Fab and anti-CD28 Fab from the oligomer streptavidin reagent.

After addition of biotin, the cells were further incubated at 37°C for approximately an additional 24 hours. The cells were then split into two subsets as above. In the first subset, the cells were directly formulated with cryoprotectant. In the second subset, the cells were further incubated for expansion by culturing in static culture with medium exchange at 37°C for an additional 5 days, and then formulated with cryoprotectant.

C. Cell recovery and phenotypic evaluation The yields of CD3+, CD4+ and CD8+ T cells released from the column after on-column selection and stimulation were compared to cells released from the column in an alternative process in which only selection was performed on the column, including the addition of biotin to release the cells. As shown in FIG. 8A, the CD3+ T cell yield, as well as the CD4+ and CD8+ T cell yields, were similar in both processes. The total cell number and percentage of viable cells after stimulation in the cells released from the column after on-column selection and stimulation were compared to the cells after individual stimulation of selected cells in the alternative process. FIGS. 8B and 8C show the total cell number and percentage of viable cells after stimulation for both processes, respectively. In this experiment, cell recovery was greater for the process using on-column stimulation.

For processes involving an additional 5 days of culture, the quality and phenotype of the engineered cell population from each process was evaluated on day 5 of culture (day 8 from the start of the process). Figures 9A and 9B show the percentage of viable T cells recovered and the percentage of viable cells expressing an exemplary CAR (CAR+) for each process, respectively. Samples from compositions generated from both processes after 5 days of additional culture (day 8 from the start of the process) were evaluated by flow cytometry for surface expression of markers including CD4, CD8, CD27 and CCR7. Figure 9C provides a comparison of the percentage of CD4+ T cells obtained from the selection step (alternative process) or the integrated selection and stimulation step (on-column stimulation) to the percentage of viable CD4+ T cells present in the cell composition on the final day of culture after additional culture (day 5).

Figure 9D shows that the percentages of CD27-CCR7- T cells, CD27+CCR7- T cells, CD27+CCR7+ T cells, and CD27-CCR7+ T cells in the composition generated by the on-column stimulation process were similar to the percentages present in the cell composition made by the alternative process.

D. Evaluation of the Function of Engineered T Cells Produced Using On-Column Stimulation The functional capacity of engineered T cells produced using a process involving on-column stimulation was evaluated both in vitro and in vivo. Results were compared against engineered T cells produced using alternative processes.

1. In Vitro Functional Analysis Cytolytic activity was assessed by co-culturing engineered anti-CD19 CAR T cells with HEK cells expressing CD19 (HEK CD19) at an effector:target ratio of 5:1. Cytolysis was measured by impedance measurements performed at multiple time points during culture. Control conditions included incubation of T cells expressing an alternative CAR against a different target (BCMA) (HEK CD19+BCMA CAR), target cells only (HEK CD19+ only), or non-target cells (HEK CD19-&CD19 CAR) incubated with anti-CD19 CAR T cells. As shown in Figure 10, cytolytic activity was specific to target cells, and CAR T cells produced by the on-column stimulation process exhibited potent cytolytic activity similar to cells engineered by the alternative process. These data demonstrate that engineered T cells produced using on-column stimulation have comparable efficacy to engineered T cells produced using the alternative process.

Cytokine activity of engineered T cells was evaluated by monitoring cytokine accumulation after antigen-specific stimulation with a target cell line (CD19+ HEK cells) in the presence of a Golgi inhibitor. Cytokine production was assessed by flow cytometry after intracellular cytokine staining for surface CD4, CD8, or intracellular IFNg, IL-2, and TNF-alpha that were also co-stained for anti-CD19 CAR. Figures 11A-11C show the percentages of CD4+ and CD8+ T cell subsets from each manufacturing process that express IFNg, IL-2, and TNF-alpha, respectively. T cells were produced according to the two methods, while T cells lacking CAR expression were used as a control. These data demonstrate that CAR T cell antigen-specific cytokine production is comparable in cells generated between manufacturing processes.

2. In Vivo Functional Analysis The anti-tumor activity of T cell compositions containing anti-CD19 CAR T cells generated from on-column stimulation and alternative engineering processes was compared in vivo.

Immunocompromised NSG mice were injected (iv) with ^5x105 B cell lymphoma cell line (Raji) on day 0. On day 7, mice were injected with ^0.75x106 CAR+ T cells from CAR+ engineered compositions generated by either on-column stimulation or the alternative process. Three manufacturing runs were completed for each process from three different donors and each engineered CAR+ T therapeutic cell composition generated was tested. Figures 12A-12C show the CD4:CD8 ratio, transduction efficiency, and percentage of viable cells, respectively, of each engineered therapeutic composition prior to injection. As shown, cells from both processes produced comparable engineered cells for each donor, although some donor variation was observed.

Tumor burden was measured in vivo by in-vivo luminescence imaging at different time points up to 41 days after administration of CAR-T cells. Six days after tumor injection, animals within all treatment groups showed similar tumor burden (Figure 13). As shown in Figure 14, tumor burden was substantially reduced over time across all treatment groups. These results demonstrated comparable anti-tumor efficacy between CAR+ engineered therapeutic T cells generated by the manufacturing process.

E. Conclusion Taken together, these data show that on-column selection and stimulation can be used in a process for generating engineered T cells (e.g., CAR+ T cells), including a process in which selected T cells are harvested from the column within 4.5 hours after initiation of stimulation with anti-CD3/anti-CD28 oligomer reagents for use in downstream steps of the process, including transduction. The results demonstrate that the on-column selection and stimulation process results in engineered (e.g., CAR+ cell) cell compositions that exhibit equivalent phenotypic and functional characteristics as alternative processes, yet can be performed more efficiently and in less time due to the ability to combine selection and stimulation in a single step.

The specific embodiments disclosed are provided for the purpose of illustrating, for example, various aspects of the invention, and are not intended to limit the scope of the invention to those specific embodiments. Various modifications of the compositions and methods described will become apparent from the description and teachings herein. Such variations can be made without departing from the true scope and spirit of the disclosure and are intended to be encompassed within the scope of the invention.

array

Claims (58)

A method for on-column stimulation of T cells, comprising the steps of:
(a) adding an oligomeric stimulatory reagent capable of delivering a stimulatory signal in T cells to a stationary phase comprising a plurality of T cells immobilized on the stationary phase, thereby initiating incubation of the oligomeric stimulatory reagent with the plurality of T cells,
the stationary phase is contained in a chromatography column and comprises a selection substance that specifically binds to a selection marker on the surface of the plurality of T cells or a subset thereof, and specific binding of the selection substance to the selection marker expressed by the one or more T cells immobilizes the one or more T cells on the stationary phase;
the oligomeric stimulatory reagent comprises one or more stimulatory agents including (i) a first stimulatory agent that is an anti-CD3 antibody or antibody fragment, and (ii) a second stimulatory agent that is an anti-CD28 antibody or antibody fragment;
process; and
(b) harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours from the start of incubation, without the addition of a competitor or free binding agent to elute one or more of the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells. A method for on-column stimulation of T cells, comprising the steps of:
(a) adding an oligomeric stimulation reagent to a stationary phase, the oligomeric stimulation reagent comprising one or more stimuli including (i) a first stimuli that is an anti-CD3 antibody or antibody fragment, and (ii) a second stimuli that is an anti-CD28 antibody or antibody fragment;
( b ) incubating the plurality of T cells immobilized on the stationary phase with one or more stimulatory agents to deliver a stimulatory signal in one or more T cells of the plurality of T cells, wherein the stationary phase comprises a selection agent contained in a chromatography column and that specifically binds to a selection marker on the surface of the one or more T cells, and wherein specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes the one or more T cells on the stationary phase; and ( c ) collecting the plurality of T cells from the stationary phase by gravity flow within 24 hours from the start of incubation, without the addition of a competitor or free binding agent to elute the one or more T cells from the stationary phase, thereby producing a composition comprising stimulated T cells. A method for on-column stimulation of T cells, comprising the steps of:
(a) applying a sample comprising a plurality of T cells to a stationary phase contained in a chromatography column, the stationary phase comprising a selection agent that binds to a selection marker on the surface of one or more T cells of the plurality of T cells, thereby immobilizing the one or more of the plurality of T cells on the stationary phase;
(b ) adding to the stationary phase an oligomeric stimulating reagent comprising one or more stimulating agents comprising (i) a first stimulating agent that is an anti-CD3 antibody or antibody fragment, and (ii) a second stimulating agent that is an anti-CD28 antibody or antibody fragment, thereby initiating incubation of the oligomeric stimulating reagent with one or more of the plurality of T cells; and (c) within 24 hours of initiating incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without the addition of a competitor or free binding agent to elute one or more of the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells. 4. The method of claim 3, wherein the initiation of incubation with the stimulating reagent is performed within 10 minutes, within 20 minutes, within 30 minutes, within 45 minutes, within 60 minutes, within 90 minutes, or within 120 minutes after adding the sample containing the plurality of T cells to the stationary phase. A method for on-column stimulation of T cells, comprising the steps of:
(1) combining (a) a sample comprising a plurality of T cells and (b) a stationary phase comprising a selection substance capable of specifically binding to a selection marker expressed on the surface of one or more of the plurality of T cells, the stationary phase being comprised in a chromatography column, and wherein specific binding of the selection substance to the selection marker immobilizes the one or more of the plurality of T cells to the stationary phase;
(2) adding an oligomeric stimulating reagent to the stationary phase, the oligomeric stimulating reagent comprising one or more stimulating agents comprising (i) a first stimulating agent that is an anti-CD3 antibody or antibody fragment, and (ii) a second stimulating agent that is an anti-CD28 antibody or antibody fragment, thereby initiating incubation of the oligomeric stimulating reagent with the one or more T cells; and (3) within 24 hours of initiating incubation, collecting one or more of the plurality of T cells from the stationary phase by gravity flow without the addition of a competitor or free binding agent to elute one or more of the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells. The method of any one of claims 1 to 5 , wherein the oligomeric stimulation reagent comprises a plurality of streptavidin or streptavidin mutein molecules, and the size of the oligomeric stimulation reagent comprises at least 100 streptavidin tetramers or streptavidin mutein tetramers. A method for on-column stimulation of T cells, comprising the steps of:
(a) adding an oligomeric stimulating reagent to a stationary phase comprising a plurality of T cells immobilized on the stationary phase, thereby initiating incubation of the stimulating reagent with one or more T cells of the plurality of T cells,
the stationary phase is contained in a chromatography column and comprises a selection agent that specifically binds to a selection marker on the surface of one or more T cells, and specific binding of the selection agent to the selection marker expressed by the one or more T cells immobilizes the one or more T cells to the stationary phase;
the oligomeric stimulating reagent comprises one or more stimulating agents including (i) a plurality of streptavidin or streptavidin mutein molecules, (ii) a first stimulating agent that is an anti-CD3 antibody or antibody fragment, and (iii) a second stimulating agent that is an anti-CD28 antibody or antibody fragment , and the size of the oligomeric stimulating reagent is (i) a radius of greater than 50 nm, (ii) a molecular weight of at least 5×10 ⁶ g/mol, and/or (iii) comprises at least 100 streptavidin or streptavidin mutein tetramers;
Process;
(b) harvesting one or more of the plurality of T cells from the stationary phase by gravity flow within 24 hours from the start of incubation, without the addition of a competitor or free binding agent to elute one or more of the plurality of T cells from the stationary phase, thereby producing a composition comprising stimulated T cells. 8. The method of claim 6 or 7 , wherein the streptavidin or streptavidin mutein molecule reversibly binds to biotin, a biotin analogue or a streptavidin-binding peptide. The streptavidin-binding peptide is
Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7),
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8),
Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:15),
SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO:16),
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17),
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19), and
The method of claim 8, wherein the mutein is selected from the group consisting of streptavidin muteins comprising the amino acid sequence ^Ile44 - ^Gly45 - ^Ala46 - ^Arg47 or Val44 ^{- Thr45} - ^Ala46 - ^Arg47 at sequence positions corresponding to positions 44 to 47 in streptavidin based on the positions in the amino acid sequence shown in SEQ ID NO:1 . 10. The method of claim 9 , wherein the streptavidin mutein comprises an amino acid sequence set forth in any of SEQ ID NOs: 3-6, 27-28, and 104-105. the selected agent comprises an agent selected from the group consisting of an antibody , an antibody fragment, a proteinaceous binding molecule with immunoglobulin-like function, an Ig domain-containing molecule, a cytokine, a chemokine, an aptamer, an MHC molecule, an MHC-peptide complex, a receptor ligand, and a binding fragment of any of the foregoing;
The method according to any one of claims 2 to 10 . The method of any one of claims 1 to 11 , wherein one or more stimulatory and/or selection agents independently comprise a monovalent antibody fragment. The method of claim 12 , wherein the monovalent antibody fragment is selected from the group consisting of a Fab fragment, an Fv fragment and a single-chain Fv fragment (scFv). The method of any one of claims 1 to 13 , wherein the first stimulatory agent is an anti-CD3 Fab and the second stimulatory agent is an anti-CD28 Fab. Harvesting one or more of the T cells from the stationary phase may be performed within 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, 18-24, 19-24, 20-24, 21-24, 22-24, 23-24, 24-24, 25-24, 26-24, 27-24, 28-24, 29-30, 31-32, 32-34, 33-36, 34-38, 35-38, 36-38, 37-38, 38-39, 39-40, 41-42, 42-44, 43-44, 44-46, 45-48, 46-48, 47-49, 48-50, 49-51, 52-53, 54-55, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 70-71, 72-72, 73-74, 74-75, 75-76, 76-77, 77-78, 78-79, 79-80, 80 15. The method of any one of claims 1 to 14, wherein the method is performed within 4, 21 to 24, 22 to 24, 23 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15 , 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6 , 2 to 5, 2 to 4 or 2 to 3 hours. 16. The method of any one of claims 1-15 , wherein the T cells are derived from a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product. 17. The method of claim 16 , wherein the T cells are derived from a previously cryofrozen apheresis product or a leukapheresis product. one or more stimulatory and/or selective agents are biotin analogues that reversibly bind to biotin, streptavidin or avidin;
Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 7),
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8),
Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 15),
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 17),
SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16),
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18), and
Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19)
18. The method of any one of claims 1 to 17, further comprising a streptavidin-binding peptide selected from the group consisting of: a calmodulin-binding peptide that reversibly binds to calmodulin; a FLAG peptide that reversibly binds to an antibody that binds to the FLAG peptide; or an oligohistidine tag that reversibly binds to an antibody that binds to the oligohistidine tag. the selection marker is a T cell coreceptor;
the selectable marker is a member of the T cell antigen receptor complex;
The selection marker is the CD3 chain;
The selection marker is CD3 zeta chain;
The selection marker is CD8;
The selection marker is CD4;
The selection marker is CD45RA;
The selection marker is CD27;
the selection marker is CD28; and/or the selection marker is CCR7;
19. The method of any one of claims 1 to 18 . the selection marker is selected from the group consisting of CD3, CD4 and CD8;
20. The method of any one of claims 1 to 19 . The method according to any one of claims 1 to 20 , wherein the specific binding between the selection agent and the selection marker does not induce a signal to the T cell or does not induce a stimulatory, activating and proliferative signal to the T cell. The method of any one of claims 1 to 21 , wherein the selection agent is an anti-CD3 Fab, an anti-CD8 Fab or an anti-CD4 Fab. the oligomeric stimulating agent is soluble;
the oligomeric stimulating reagent is not bound to a solid support, and/or the oligomeric stimulating reagent is flexible, does not contain a metallic or magnetic core, is composed entirely or primarily of organic polymers, and/or is not rigid;
23. The method of any one of claims 1 to 22 . The oligomer stimulating agent is
A radius between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm, inclusive;
a molecular weight of 5×10 ⁷ g/mol to 5×10 ⁸ g/mol, 1×10 ⁸ g/mol to 5×10 ⁸ g/mol, or 1×10 ⁸ g/mol to 2×10 ⁸ g/mol, and/or
24. The method of any one of claims 1 to 23, comprising from 1,000 to 20,000 streptavidin tetramers or streptavidin mutein tetramers, from 1,000 to 10,000 streptavidin tetramers or streptavidin mutein tetramers, or from 2,000 to 5,000 streptavidin tetramers or streptavidin mutein tetramers. 25. The method of any one of claims 1 to 24 , wherein the oligomeric stimulation reagent is added to the stationary phase at a concentration of 1 μg/million cells to 2 μg/million cells. 26. The method of any one of claims 1 to 25 , wherein the selection substance is indirectly bound to the stationary phase via a selection reagent to which it reversibly binds. 27. The method of claim 26, wherein the selection reagent comprises streptavidin; avidin; a mutein of streptavidin that reversibly binds to biotin or a biotin analog; an avidin or a mutein of streptavidin that reversibly binds to a streptavidin-binding peptide; a reagent comprising at least two chelating groups, the at least two chelating groups being capable of binding to a transition metal ion; a substance capable of binding to an oligohistidine affinity tag; a substance capable of binding to glutathione-S-transferase; calmodulin or an analog thereof; a substance capable of binding to calmodulin-binding peptide (CBP); a substance capable of binding to a FLAG peptide; a substance capable of binding to an HA tag; a substance capable of binding to maltose binding protein (MBP); a substance capable of binding to an HSV epitope; a substance capable of binding to a myc epitope; or a substance capable of binding to a biotinylated carrier protein. Incubating with one or more stimuli releases one or more of the plurality of immobilized T cells from the stationary phase, and/or collecting by gravity flow comprises adding medium to the stationary phase that does not contain a competitor or free binding agent for eluting the T cells from the stationary phase, and/or the competitor or free binding agent comprises biotin or a biotin analogue.
28. The method of any one of claims 1 to 27 . 29. The method of any one of claims 1 to 28 , wherein the competitor or free binder comprises D-biotin. 30. The method of any one of claims 1 to 29 , further comprising the step of introducing into a T cell of said composition comprising stimulated T cells a recombinant nucleic acid molecule encoding a recombinant protein. 31. The method of claim 30 , wherein the recombinant protein is a chimeric antigen receptor, thereby generating a composition comprising engineered T cells. 32. The method of claim 31 , wherein the composition comprises a transduced T cell. The method of any one of claims 30 to 32 , wherein the introduction of the recombinant nucleic acid is achieved by transduction with a viral particle. 34. The method of claim 33 , wherein the viral particle is a retroviral vector particle. 35. The method of claim 34 , wherein the viral particle is a lentiviral particle. The method of any one of claims 30-34 , further comprising culturing the composition comprising the engineered T cells under conditions for viral integration. 37. The method of claim 36 , further comprising culturing the composition comprising the engineered T cells at a temperature of 37°±2° C. 38. The method of claim 36 or 37 , wherein the step of culturing the composition comprising the engineered T cells is carried out for at least 18 hours and up to 96 hours, up to 72 hours, up to 48 hours, or up to 24 hours after introduction. The method of any one of claims 30-38 , further comprising culturing the composition comprising the engineered T cells under conditions for expanding the T cells. 40. The method of claim 39 , wherein the culturing is carried out for a period of time not longer than 14 days, not longer than 12 days, not longer than 10 days, not longer than 8 days, not longer than 6 days, or not longer than 5 days. The method of any one of claims 30-40 , further comprising harvesting the engineered T cells, thereby generating an output population of engineered T cells. 42. The method of claim 41 , wherein the engineered T cells are harvested at a time point between 48 and 120 hours, inclusive, after initiation of exposure to the oligomeric stimulating reagent. The method of claim 41 or 42, wherein the percentage of naive-like cells is greater than 60% of the total T cells in the population, the total CD4+ T cells in the population, the total CD8+ T cells in the population, or any of the preceding recombinant protein -expressing cells in the population at the time of harvest. The method of claim 43 , wherein the naive-like T cells comprise CD27+CCR7+ cells. 45. The method of any one of claims 41-44 , further comprising formulating the harvested cells for cryopreservation and/or administration to a subject. 46. The method of any one of claims 41-45, further comprising formulating the harvested cells in the presence of a pharma- ceutically acceptable excipient or cryoprotectant for cryopreservation and/or administration to a subject. The method of any one of claims 30 to 46 , wherein the introducing, incubating and/or culturing steps are carried out in serum-free medium. 48. The method of claim 47 , wherein the serum-free medium does not contain recombinant human IL-2, recombinant human IL-15 or recombinant human IL-7. The method of any one of claims 30 to 48 , further comprising washing the T cells. 50. The method of claim 49 , wherein the washing step reduces or removes the oligomeric stimulating reagent and/or one or more stimulating substances in the composition. the T cells comprise antigen-specific T cells or a population thereof, helper T cells or a population thereof, cytotoxic T cells or a population thereof, memory T cells or a population thereof, or regulatory T cells or a population thereof; and/or
the T cells comprise CD3+ T cells or comprise CD4+ and/or CD8+ T cells;
The method of any one of claims 1 to 50 . selecting a T cell subset from the stimulated T cells of said composition, followed by the introduction of any one of claims 30 to 51 , to introduce the recombinant nucleic acid molecule into said selected T cell subset;
selecting a T cell subset from a composition comprising engineered T cells, followed by the incubation of any one of claims 36 to 51 , wherein said selected T cell subset is incubated under conditions for viral integration;
comprising a step of selecting a T cell subset from a composition comprising engineered T cells, followed by a culturing step according to any one of claims 26 to 51 , wherein said selected T cell subset is cultured under conditions for expanding the T cells; and/or comprising a step of selecting a T cell subset from a composition comprising engineered T cells, followed by a harvesting step according to any one of claims 41 to 51 , wherein said selected T cell subset is harvested to generate an output population of engineered T cells.
52. The method of any one of claims 1 to 51 . 53. The method of claim 52 , wherein the selecting step is carried out by affinity column chromatography. The method of claim 52 or 53, wherein the T cell subset is a naive-like T cell; is a T cell that is surface positive for a marker expressed on a naive-like T cell; is CCR7+CD45RA+, CD27+ CCR7 +, or CD62L-CCR7+; and/or expresses a recombinant protein. The stationary phase comprises a chromatographic matrix;
The stationary phase is a non-magnetic or non-magnetizable material;
the stationary phase has a binding capacity of 75 to 125 million T cells per mL of stationary phase; and/or the stationary phase is 20 mL;
55. The method of any one of claims 1 to 54 . 56. The method of claim 55 , wherein the stationary phase has a binding capacity of 2 billion ± 500 million cells. 57. The method of claim 55 or 56 , wherein the chromatography matrix is for column chromatography. The method of any one of claims 1 to 57 , wherein the method is performed automatically.

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